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Cornelia de Lange syndrome; Genetic disorder; Intragenic duplication Síndrome de Cornelia de Lange; Trastorno genético; Duplicación intragénica Síndrome de Cornelia de Lange; Trastorn genètic; Duplicació intragènica Cornelia de Lange syndrome (CdLS) is a multisystemic genetic disorder characterized by distinctive facial features, growth retardation, and intellectual disability, as well as various systemic conditions. It is caused by genetic variants in genes related to the cohesin complex. Single-nucleotide variations are the best-known genetic cause of CdLS; however, copy number variants (CNVs) clearly underlie a substantial proportion of cases of the syndrome. The NIPBL gene was thought to be the locus within which clinically relevant CNVs contributed to CdLS. However, in the last few years, pathogenic CNVs have been identified in other genes such as HDAC8, RAD21, and SMC1A. Here, we studied an affected girl presenting with a classic CdLS phenotype heterozygous for a de novo ~32 kbp intragenic duplication affecting exon 10 of HDAC8. Molecular analyses revealed an alteration in the physiological splicing that included a 96 bp insertion between exons 9 and 10 of the main transcript of HDAC8. The aberrant transcript was predicted to generate a truncated protein whose accessibility to the active center was restricted, showing reduced ease of substrate entry into the mutated enzyme. Lastly, we conclude that the duplication is responsible for the patient’s phenotype, highlighting the contribution of CNVs as a molecular cause underlying CdLS. This work was supported by the Spanish Ministry of Health-ISCIII Fondo de Investigación Sanitaria (FIS) (Ref. PI19/01860, to F.J.R. and J.P.) and Diputación General de Aragón-FEDER: European Social Fund (Grupo de Referencia B32_17R/B32_20R, to J.P.). A.L.-P. is supported by a “Juan de la Cierva-Incorporación” postdoctoral grant from MICIU (Spanish Ministry of Science and Universities), M.G.-S. is supported by a Predoctoral Fellowship from the Diputación General de Aragón, and C.L.-C. is supported by a Predoctoral Fellowship from the MH-ISCIII. This work was also supported by Spanish government grants RTI2018-094434-B-I00 (MCIU/AEI/FEDER, UE) and DTS20-00024 (ISCIII) to P.G.-P., as well as funds from the European JPIAMR network “EPIC-Alliance” to P.G.-P. The computational support of the “Centro de Computación Científica CCC-UAM” is gratefully recognized. This work was also partially supported by Spanish Instituto de Salud Carlos III, Fondo de Investigaciones Sanitarias co-funded with ERDF funds, Grant No. FIS PI20/01767) to A.P. and by Spanish Instituto de Salud Carlos III, Fondo de Investigaciones Sanitarias co-funded with ERDF funds, Grant No. FIS PI18/000687 to E.F.T.

Schuurs–Hoeijmakers syndrome (SHMS) or PACS1 Neurodevelopmental disorder is a rare disorder characterized by intellectual disability, abnormal craniofacial features and congenital malformations. SHMS is an autosomal dominant hereditary disease caused by pathogenic variants in the PACS1 gene. PACS1 is a trans-Golgi-membrane traffic regulator that directs protein cargo and several viral envelope proteins. It is upregulated during human embryonic brain development and has low expression after birth. So far, only 54 patients with SHMS have been reported. In this work, we report on seven new identified SHMS individuals with the classical c.607C > T: p.Arg206Trp PACS1 pathogenic variant and review clinical and molecular aspects of all the patients reported in the literature, providing a summary of clinical findings grouped as very frequent (≥75% of patients), frequent (50–74%), infrequent (26–49%) and rare (less than ≤25%), This work was possible thanks to the funding provided by the project “Proyecto Piloto para la mejora del diagnóstico genético en personas y familias afectadas o con sospecha de padecer enfermedades raras de base genética” of the Ministry of Health, under the grant BOCM-20181126-24 provided by the Consejería de Sanidad de la Comunidad de Madrid. Funding to J.P. and F.J.R. was partially provided by the group research grant DGA/FEDER B32_17R/B32_20R

Postzygotic mosaicism (PZM) in NIPBL is a strong source of causality for Cornelia de Lange syndrome (CdLS) that can have major clinical implications. Here, we further delineate the role of somatic mosaicism in CdLS by describing a series of 11 unreported patients with mosaic disease-causing variants in NIPBL and performing a retrospective cohort study from a Spanish CdLS diagnostic center. By reviewing the literature and combining our findings with previously published data, we demonstrate a negative selection against somatic deleterious NIPBL variants in blood. Furthermore, the analysis of all reported cases indicates an unusual high prevalence of mosaicism in CdLS, occurring in 13.1% of patients with a positive molecular diagnosis. It is worth noting that most of the affected individuals with mosaicism have a clinical phenotype at least as severe as those with constitutive pathogenic variants. However, the type of genetic change does not vary between germline and somatic events and, even in the presence of mosaicism, missense substitutions are located preferentially within the HEAT repeat domain of NIPBL. In conclusion, the high prevalence of mosaicism in CdLS as well as the disparity in tissue distribution provide a novel orientation for the clinical management and genetic counselling of families., Spanish Ministry of Health-ISCIII Fondo de Investigación Sanitaria (FIS) [Ref. PI19/01860, to F.J.R. and J.P.]; Diputación General de Aragón-FEDER: European Social Fund [Grupo de Referencia B32_17R / B32_20R, to J.P.]. A.L-P is supported by a “Juan de la Cierva-Incorporación” postdoctoral grant from MICIU (Spanish Ministry of Science and Universities)

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The gamma-1 isoform of casein kinase 1, the protein encoded by CSNK1G1, is involved in the growth and morphogenesis of cells. This protein is expressed ubiquitously among many tissue types, including the brain, where it regulates the phosphorylation of N-methyl-D-aspartate receptors and plays a role in synaptic transmission. One prior individual with a de novo variant in CSNK1G presenting with severe developmental delay and early-onset epilepsy has been reported. Here we report an updated clinical history of this previously published case, as well as four additional individuals with de novo variants in CSNK1G1 identified via microarray-based comparative genomic hybridization, exome, or genome sequencing. All individuals (n = 5) had developmental delay. At least three individuals had diagnoses of autism spectrum disorder. All participants were noted to have dysmorphic facial features, although the reported findings varied widely and therefore may not clearly be recognizable. None of the participants had additional major malformations. Taken together, our data suggest that CSNK1G1 may be a cause of syndromic developmental delay and possibly autism spectrum disorder.

Cornelia de Lange syndrome (CdLS) is an archetypical genetic syndrome that is characterized by intellectual disability, well-defined facial features, upper limb anomalies and atypical growth, among numerous other signs and symptoms. It is caused by variants in any one of seven genes, all of which have a structural or regulatory function in the cohesin complex. Although recent advances in next-generation sequencing have improved molecular diagnostics, marked heterogeneity exists in clinical and molecular diagnostic approaches and care practices worldwide. Here, we outline a series of recommendations that document the consensus of a group of international experts on clinical diagnostic criteria, both for classic CdLS and non-classic CdLS phenotypes, molecular investigations, long-term management and care planning., Cornelia de Lange syndrome is a genetic disorder affecting multiple organ systems that exhibits great phenotypic heterogeneity. This Consensus Statement summarizes recommendations for the diagnosis and management of patients with Cornelia de Lange syndrome.

RAD21 encodes a key component of the cohesin complex, and variants in RAD21 have been associated with Cornelia de Lange Syndrome (CdLS). Limited information on phenotypes attributable to RAD21 variants and genotype–phenotype relationships is currently published. We gathered a series of 49 individuals from 33 families with RAD21 alterations [24 different intragenic sequence variants (2 recurrent), 7 unique microdeletions], including 24 hitherto unpublished cases. We evaluated consequences of 12 intragenic variants by protein modelling and molecular dynamic studies. Full clinical information was available for 29 individuals. Their phenotype is an attenuated CdLS phenotype compared to that caused by variants in NIPBL or SMC1A for facial morphology, limb anomalies, and especially for cognition and behavior. In the 20 individuals with limited clinical information, additional phenotypes include Mungan syndrome (in patients with biallelic variants) and holoprosencephaly, with or without CdLS characteristics. We describe several additional cases with phenotypes including sclerocornea, in which involvement of the RAD21 variant is uncertain. Variants were frequently familial, and genotype–phenotype analyses demonstrated striking interfamilial and intrafamilial variability. Careful phenotyping is essential in interpreting consequences of RAD21 variants, and protein modeling and dynamics can be helpful in determining pathogenicity. The current study should be helpful when counseling families with a RAD21 variation., Spanish Ministry of Science, Innovation and Universities/State Research Agency RTC-2017-6494-1 and RTI2018-094434-B-I00 (MCIU/AEI/FEDER, UE) as well as funds from the European JPIAMR-VRI network “CONNECT” to PG-P

Cornelia de Lange syndrome (CdLS), Rubinstein-Taybi syndrome (RSTS), and KBG syndrome are three distinct developmental human disorders. Variants in seven genes belonging to the cohesin pathway, NIPBL, SMC1A, SMC3, HDAC8, RAD21, ANKRD11, and BRD4, were identified in about 80% of patients with CdLS, suggesting that additional causative genes remain to be discovered. Two genes, CREBBP and EP300, have been associated with RSTS, whereas KBG results from variants in ANKRD11. By exome sequencing, a genetic cause was elucidated in two patients with clinical diagnosis of CdLS but without variants in known CdLS genes. In particular, genetic variants in EP300 and ANKRD11 were identified in the two patients with CdLS. EP300 and ANKRD11 pathogenic variants caused the reduction of the respective proteins suggesting that their low levels contribute to CdLS-like phenotype. These findings highlight the clinical overlap between CdLS, RSTS, and KBG and support the notion that these rare disorders are linked to abnormal chromatin remodeling, which in turn affects the transcriptional machinery.

Cornelia de Lange Syndrome (CdLS) is an autosomal dominant (NIPBL, SMC3, and RAD21) or X-linked (SMC1A and HDAC8) disorder, characterized by distinctive craniofacial appearance, growth retardation, intellectual disability, and limb anomalies. In 2005, the Spanish CdLS Reference Center was started and now we have more than 270 cases in our database. In this special issue, we describe some of the unique or atypical patients studied by our group, whose clinical features have contributed to the expansion of the CdLS classical phenotype, helping clinicians to diagnose it. We include the case of a male with unilateral tibial hypoplasia and peroneal agenesis who had a mutation in NIPBL; we also describe one patient with a mutation in NIPBL and somatic mosaicism identified by new generation sequencing techniques; we also include one patient with CdLS and Turner syndrome; and last, an interesting patient with a duplication of the SMC1A gene. Finally, we make a short review of the splicing mutations we have found in NIPBL regarding the new knowledge on the physiological variants of the gene. © 2016 Wiley Periodicals, Inc.

Cornelia de Lange syndrome (CdLS) is a rare developmental disorder caused by mutations in genes related to the cohesin complex. For its association with chromatin, cohesin depends on a heterodimer formed by NIPBL and MAU2, which interact via their respective N-termini. Variants in NIPBL are the main cause of CdLS and result in NIPBL haploinsufficiency.Using CRISPR, we generated cells homozygous for an out-of-frame duplication in NIPBL. Remarkably, alternative translation initiation rescued NIPBL expression in these cells and produced an N-terminally truncated NIPBL that lacks MAU2-interaction domain, causing a dramatic reduction of MAU2 protein levels. Strikingly, this protective mechanism allows nearly normal amounts of cohesin to be loaded onto chromatin in a manner that is independent of functional NIPBL/MAU2 complexes and therefore in contrast to previous findings.We also report the first pathogenic variant in MAU2, a deletion of seven amino acids important for wrapping the N-terminus of NIPBL within MAU2. The mutation causes dramatic reduction of MAU2 heterodimerization with NIPBL, hence undermining the stability of both proteins.Our data confirm NIPBL haploinsufficiency as the major pathogenic mechanism of CdLS and give new insights into the molecular mechanisms responsible for this neurodevelopmental disorder. Our work also unveils an alternative translation-based mechanism that protects cells from out-of-frame variants of NIPBL and that may be of relevance in other genetic conditions.

Cornelia de Lange syndrome (CdLS) is a clinically heterogeneous disorder characterized by typical facial dysmorphism, cognitive impairment and multiple congenital anomalies. Approximately 75% of patients carry a variant in one of the five cohesin-related genes NIPBL, SMC1A, SMC3, RAD21 and HDAC8. Herein we report on the clinical and molecular characterization of 11 patients carrying 10 distinct variants in HDAC8. Given the high number of variants identified so far, we advise sequencing of HDAC8 as an indispensable part of the routine molecular diagnostic for patients with CdLS or CdLS-overlapping features. The phenotype of our patients is very broad, whereas males tend to be more severely affected than females, who instead often present with less canonical CdLS features. The extensive clinical variability observed in the heterozygous females might be at least partially associated with a completely skewed X-inactivation, observed in seven out of eight female patients. Our cohort also includes two affected siblings whose unaffected mother was found to be mosaic for the causative mutation inherited to both affected children. This further supports the urgent need for an integration of highly sensitive sequencing technology to allow an appropriate molecular diagnostic, genetic counseling and risk prediction.

Item does not contain fulltext SMC1A encodes one of the proteins of the cohesin complex. SMC1A variants are known to cause a phenotype resembling Cornelia de Lange syndrome (CdLS). Exome sequencing has allowed recognizing SMC1A variants in individuals with encephalopathy with epilepsy who do not resemble CdLS. We performed an international, interdisciplinary study on 51 individuals with SMC1A variants for physical and behavioral characteristics, and compare results to those in 67 individuals with NIPBL variants. For the Netherlands all known individuals with SMC1A variants were studied, both with and without CdLS phenotype. Individuals with SMC1A variants can resemble CdLS, but manifestations are less marked compared to individuals with NIPBL variants: growth is less disturbed, facial signs are less marked (except for periocular signs and thin upper vermillion), there are no major limb anomalies, and they have a higher level of cognitive and adaptive functioning. Self-injurious behavior is more frequent and more severe in the NIPBL group. In the Dutch group 5 of 13 individuals (all females) had a phenotype that shows a remarkable resemblance to Rett syndrome: epileptic encephalopathy, severe or profound intellectual disability, stereotypic movements, and (in some) regression. Their missense, nonsense, and frameshift mutations are evenly spread over the gene. We conclude that SMC1A variants can result in a phenotype resembling CdLS and a phenotype resembling Rett syndrome. Resemblances between the SMC1A group and the NIPBL group suggest that a disturbed cohesin function contributes to the phenotype, but differences between these groups may also be explained by other underlying mechanisms such as moonlighting of the cohesin genes.

International audience; Disorders/differences of sex development (DSD) cause profound psychological and reproductive consequences for the affected individuals, however, most are still unexplained at the molecular level. Here, we present a novel gene, 3-hydroxy-3-methylglutaryl coenzyme A synthase 2 (HMGCS2), encoding a metabolic enzyme in the liver important for energy production from fatty acids, that shows an unusual expression pattern in developing fetal mouse gonads. Shortly after gonadal sex determination it is up-regulated in the developing testes following a very similar spatial and temporal pattern as the male-determining gene Sry in Sertoli cells before switching to ovarian enriched expression. To test if Hmgcs2 is important for gonad development in mammals, we pursued two lines of investigations. Firstly, we generated Hmgcs2-null mice using CRISPR/Cas9 and found that these mice had gonads that developed normally even on a sensitized background. Secondly, we screened 46,XY DSD patients with gonadal dysgenesis and identified two unrelated patients with a deletion and a deleterious missense variant in HMGCS2 respectively. However, both variants were heterozygous, suggesting that HMGCS2 might not be the causative gene. Analysis of a larger number of patients in the future might shed more light into the possible association of HMGCS2 with human gonadal development.

Formation of tubulin/Na+,K+-ATPase (NKA) complex in erythrocytes of hypertensive subjects results in a 50% reduction in NKA activity. We demonstrate here that detyrosinated tubulin, which is increased in hypertensive erythrocytes membranes, enhances the inhibitory effect of acetylated tubulin on NKA activity. Moreover, we report a reduced content and activity of the enzyme tubulin tyrosine ligase in erythrocytes of hypertensive subjects. Such alterations are related to changes in erythrocyte deformability. Our findings indicate that the detyrosination/tyrosination cycle of tubulin is important in regulation of NKA activity, and that abnormalities in this cycle are involved in hypertension development.

Cornelia de Lange syndrome (CdLS) is amultisystemgenetic disorder with distinct facies, growth failure, intellectual disability, distal limb anomalies, gastrointestinal and neurological disease. Mutations in NIPBL, encoding a cohesin regulatory protein, account for >80% of cases with typical facies. Mutations in the core cohesin complex proteins, encoded by the SMC1A, SMC3 and RAD21 genes, together account for ̃5% of subjects, often with atypical CdLS features. Recently, we identified mutations in the X-linked gene HDAC8 as the cause of a small number of CdLS cases. Here, we report a cohort of 38 individuals with an emerging spectrum of features caused by HDAC8 mutations. For several individuals, the diagnosis of CdLS was not considered prior to genomic testing. Most mutations identified are missense and de novo. Many cases are heterozygous females, each with marked skewing of X-inactivation in peripheral blood DNA.Wealso identified eight hemizygous males who are more severely affected. The craniofacial appearance caused by HDAC8 mutations overlaps that of typical CdLS but often displays delayed anterior fontanelle closure, ocular hypertelorism, hooding of the eyelids, a broader nose and dental anomalies, which may be useful discriminating features. HDAC8 encodes the lysine deacetylase for the cohesin subunit SMC3 and analysis of the functional consequences of the missense mutations indicates that all cause a loss of enzymatic function. These data demonstrate that loss-of-function mutations in HDAC8 cause a range of overlapping human developmental phenotypes, including a phenotypically distinct subgroup of CdLS. © The Author 2014. Published by Oxford University Press. All rights reserved.Published by Oxford University Press. All rights reserved.

Mitochondrial HMG-CoA synthase deficiency is a rare inherited metabolic disorder that affects ketone-body synthesis. Acute episodes include vomiting, lethargy, hepatomegaly, hypoglycaemia, dicarboxylic aciduria, and in severe cases, coma. This deficiency may have been under-diagnosed owing to the absence of specific clinical and biochemical markers, limitations in liver biopsy and the lack of an effective method of expression and enzyme assay for verifying the mutations found. To date, eight patients have been reported with nine allelic variants of the HMGCS2 gene. We present a new method of enzyme expression and a modification of the activity assay that allows, for first time, the functional study of missense mutations found in patients with this deficiency. Four of the missense mutations (p.V54M, p.R188H, p.G212R and p.G388R) did not produce proteins that could have been detected in soluble form by western blot; three produced a total loss of activity (p.Y167C, p.M307T and p.R500H) and one, variant p.F174L, gave an enzyme with a catalytic efficiency of 11.5%. This indicates that the deficiency may occur with partial loss of activity of enzyme. In addition, we describe a new patient with this deficiency, in which we detected the missense allelic variant, c.1162G>A (p.G388R) and the nonsense variant c.1270C>T (p.R424X). © 2013 Elsevier Masson SAS., Diputación General de Aragón (DGA) (Grupo Consolidado B20); European Social Fund (“Construyendo Europa desde Aragón”); Spanish Ministry of Education and Science (SAF2004-06843-C03); Spanish Ministerio de Ciencia and Ministerio de Economía y Competitividad (SAF2007-61926, IPT2011-0964-900000, SAF2011-13156-E); Spanish Instituto de Salud Carlos III (CIBER Fisiopatología de la Obesidad y Nutrición); European Commission (FP7 HEALTH-F3-2009-223431, EU project “Divinocell”, FP7 HEALTH-2011-278603; EU project “Dorian”); European Social Fund

The disorders caused by mutations in genes encoding subunits and accessory proteins of cohesin complex are collectively termed as cohesinopathies. The best known cohesinopathy is Cornelia de Lange Syndrome (CdLS), which is a multisystem developmental disorder characterized by facial dysmorphism, limb malformations, growth and cognitive impairment. Mutations in five genes, encoding subunits of the cohesin complex (SMC1A, SMC3, RAD21) and its regulators (NIPBL, HDAC8), are responsible for ∼ 70% of CdLS cases. We describe a 16-year-old boy with facial dysmorphism, growth retardation, intellectual disability, hirsutism and small hands, who has a small Supernumerary Marker Chromosome (sSMC) present in mosaic form. sSMC is composed of two duplicated segments encompassing 17 genes including SMC1A gene, at the regions Xp11.22 and Xp11.21q11.1. Clinical comparison between our patient with a previously reported individual with a SMC1A duplication and four male carriers of similar sSMC reported in databases, suggest that they all share clinical features related to cohesinopathies. Although our patient does not have the classical CdLS craniofacial phenotype, he has pre and postnatal growth retardation, intellectual disability and mild musculoskeletal anomalies, features commonly seen in patients with cohesinopathies.

WOS: 000394359900004, PubMed ID: 28120103, The coordinated tissue-specific regulation of gene expression is essential for the proper development of all organisms. Mutations in multiple transcriptional regulators cause a group of neurodevelopmental disorders termed "transcriptomopathies" that share core phenotypical features including growth retardation, developmental delay, intellectual disability and facial dysmorphism. Cornelia de Lange syndrome (CdLS) belongs to this class of disorders and is caused by mutations in different subunits or regulators of the cohesin complex. Herein, we report on the clinical and molecular characterization of seven patients with features overlapping with CdLS who were found to carry mutations in chromatin regulators previously associated to other neurodevelopmental disorders that are frequently considered in the differential diagnosis of CdLS. The identified mutations affect the methyltransferase-encoding genes KMT2A and SETD5 and different subunits of the SWI/SNF chromatin-remodeling complex. Complementary to this, a patient with Coffin-Siris syndrome was found to carry a missense substitution in NIPBL. Our findings indicate that mutations in a variety of chromatin-associated factors result in overlapping clinical phenotypes, underscoring the genetic heterogeneity that should be considered when assessing the clinical and molecular diagnosis of neurodevelopmental syndromes. It is clear that emerging molecular mechanisms of chromatin dysregulation are central to understanding the pathogenesis of these clinically overlapping genetic disorders., German Federal Ministry of Education and Research (BMBF) (CHROMATIN-Net); Spain'sMinistry of Health-ISCIII [FIS PI12-01318]; Medical Faculty of the University of Lubeck [J09-2017]; German Federal Ministry of Education and Research (BMBF) (E-Rare-2 TARGET-CdLS), This work was funded by the German Federal Ministry of Education and Research (BMBF) (CHROMATIN-Net: to F.K., G.G.-K., N.C.B. and D.W.; E-Rare-2 TARGET-CdLS: to F.J.K, K.W. and E.W.), by the Spain'sMinistry of Health-ISCIII (Ref. FIS PI12-01318) (to F. J.R. and J.P.) and by the Medical Faculty of the University of Lubeck (J09-2017 to I.P.).

Cornelia de Lange syndrome (CdLS) is a rare genetically heterogeneous disorder with a high phenotypic variability including mental retardation, developmental delay, and limb malformations. The genetic causes in about 30% of patients with CdLS are still unknown. We report on the functional characterization of two intronicNIPBLmutations in two patients with CdLS that do not affect a conserved splice-donor or acceptor site. Interestingly, mRNA analyses showed aberrantly spliced transcripts missing exon 28 or 37, suggesting the loss of the branch site by the c.5329-15A>G transition and a disruption of the polypyrimidine by the c.6344del(-13)_(-8) deletion. While the loss of exon 28 retains the reading frame of theNIBPLtranscript resulting in a shortened protein, the loss of exon 37 shifts the reading frame with the consequence of a premature stop of translation. Subsequent quantitative PCR analysis demonstrated a 30% decrease of the totalNIPBLmRNA levels associated with the frameshift transcript. Consistent with our results, this patient shows a more severe phenotype compared to the patient with the aberrant transcript that retains its reading frame. Thus, intronic variants identified by sequencing analysis in CdLS diagnostics should carefully be examined before excluding them as nonrelevant to disease.

3-hydroxy-3-methylglutaryl-CoA (HMG-CoA) lyase adopts a (betaalpha)(8) TIM barrel structure with an additional beta9, alpha11 and alpha12 helices. Location of HMG part of the substrate has been suggested but the binding mode for the CoA moiety remains to be resolved. As mutation F305 fs(-2), which involves the last 21 residues of the protein, and mutation K48N caused 3-hydroxy-3-methylglutaric aciduria in two patients, we examined the role of the C-terminal end and Lys(48) in enzyme activity. Expression studies of various C-terminal-end-deleted and K48N-mutated proteins revealed that residues 311-313 (localized in the loop between alpha11 and alpha12 helices) and Lys(48) are essential for enzyme activity. An in silico docking model locating HMG-CoA on the surface of the enzyme implicates Asn(311) and Lys(313) in substrate binding by establishing multiple polar contacts with phosphate and ribose groups of adenosine, and Lys(48) by contacting the carboxyl group of the panthotenic acid moiety.

3-Hydroxy-3-methylglutaric aciduria is a rare autosomal recessive genetic disorder that affects ketogenesis and leucine metabolism. The disease is caused by mutations in the gene coding for 3-hydroxy-3-methylglutaryl-coenzyme A lyase (HL). To date 26 different mutations have been described. A (betaalpha)(8) TIM barrel structure has been proposed for the protein, and almost all missense mutations identified so far localize in the beta sheets that define the inside cavity. We report an Italian patient who bears homozygously a novel HL mutation, c.608GA (p. G203E) in beta sheet six. A structural model of the mutated protein suggests that glutamic acid 203 impedes catalysis by blocking the entrance to the inner cavity of the enzyme. Loss of functionality has been confirmed in expression studies in E. coli, which demonstrate that the G203E mutation completely abolishes enzyme activity. Beta sheet six and beta sheet two are the two protein regions that accumulate most missense mutations, indicating their importance in enzyme functionality. A model for the mechanism of enzyme function is proposed.

Mitochondrial HMG-CoA synthase deficiency is an inherited metabolic disorder caused by a defect in the enzyme that regulates the formation of ketone bodies. Patients present with hypoketotic hypoglycaemia, encephalopathy and hepatomegaly, usually precipitated by an intercurrent infection or prolonged fasting. The diagnosis may easily be missed as previously reported results of routine metabolic investigations, urinary organic acids and plasma acylcarnitines may be nonspecific or normal, and a high index of suspicion is required to proceed to further confirmatory tests. We describe a further acute case in which the combination of urinary organic acids, low free carnitine and changes in the plasma acylcarnitine profile on carnitine supplementation were very suggestive of a defect in ketone synthesis. The diagnosis of mitochondrial HMG-CoA synthase deficiency was confirmed on genotyping, revealing two novel mutations: c.614G > A (R188H) and c.971T > C (M307T). A further sibling, in whom the diagnosis had not been made acutely, was also found to be affected. The possible effects of these mutations on enzyme activity are discussed.

Deficiency of mitochondrial 3-hydroxy-3-methylglutaryl-CoA synthase (mHMGS) is a recessive disorder of ketogenesis that has been previously diagnosed in two children with hypoglycaemic hypoketotic coma during fasting periods. Here, we report the results of molecular investigations in a third patient affected by this disease. Sequencing of the entire coding region of the HMGCS2 gene revealed two missense mutations, G212R and R500H. Mendelian inheritance was confirmed by the analysis of parental samples and neither of the mutations was found on 200 control chromosomes. Functional relevance was confirmed by in vitro expression studies in cytosolic HMGS-deficient cells. Whereas wild-type cDNA of the HMGCS2 gene reverted the auxotrophy for mevalonate, the cDNAs of the mutants did not. The disease may be recognised by specific clinical and biochemical features but it is difficult to confirm enzymatically since the gene is expressed only in liver and testis. Molecular studies may facilitate or confirm future diagnoses in affected patients.

The cohesin loading protein is required for nonhomologous end joining of double-strand DNA breaks, DNA double strand breaks (DSBs) are mainly repaired via homologous recombination (HR) or nonhomologous end joining (NHEJ). These breaks pose severe threats to genome integrity but can also be necessary intermediates of normal cellular processes such as immunoglobulin class switch recombination (CSR). During CSR, DSBs are produced in the G1 phase of the cell cycle and are repaired by the classical NHEJ machinery. By studying B lymphocytes derived from patients with Cornelia de Lange Syndrome, we observed a strong correlation between heterozygous loss-of-function mutations in the gene encoding the cohesin loading protein NIPBL and a shift toward the use of an alternative, microhomology-based end joining during CSR. Furthermore, the early recruitment of 53BP1 to DSBs was reduced in the NIPBL-deficient patient cells. Association of NIPBL deficiency and impaired NHEJ was also observed in a plasmid-based end-joining assay and a yeast model system. Our results suggest that NIPBL plays an important and evolutionarily conserved role in NHEJ, in addition to its canonical function in sister chromatid cohesion and its recently suggested function in HR.

Cornelia de Lange Syndrome (CdLS) is a congenital autosomal dominant (NIPBL, SMC3 and RAD21) or X-linked (SMC1A and HDAC8) disorder characterized by facial dysmorphism, pre and postnatal growth retardation, developmental delay and/or intellectual disability, and multiorgan involvement. Musculoskeletal malformations are usually bilateral and affect mainly the upper limbs; the range goes from brachyclinodactyly to severe reduction defects. Instead lower extremities are usually less and mildly involved. Here, we report on a 3-year-old Senegalese boy with typical craniofacial CdLS features, pre and postnatal growth retardation, atrial septal defect, developmental delay and right ipsilateral limb malformations, consistent with oligodactyly of the 3rd and 4th fingers, tibial agenesis and fibula hypoplasia. Exome sequencing and Sanger sequencing showed a novel missense mutation in NIPBL gene (c.6647A>G; p.(Tyr2216Cys)), which affects a conserved residue located within NIPBL HEAT repeat elements. Pyrosequencing analysis of NIPBL gene, disclosed similar levels of wild-type and mutated alleles in DNA and RNA samples from all tissues analyzed (oral mucosa epithelial cells, peripheral blood leukocytes and fibroblasts). These findings indicated the absence of somatic mosaicism, despite of the segmental asymmetry of the limbs, and confirmed biallelic expression for NIPBL transcripts, respectively. Additionally, conditions like Split-hand/foot malformation with long-bone deficiency secondary to duplication of BHLHA9 gene have been ruled out by the array-CGH and MLPA analysis. To our knowledge, this is the first CdLS patient described with major ipsilateral malformations of both the upper and lower extremities, that even though this finding could be due to a random event, expands the spectrum of limb reduction defects in CdLS.

Eukaryotic cells can be protected against mutations that generate stop codons by nonsense-mediated mRNA decay (NMD) and/or nonsense-associated altered splicing (NAS). However, the processes are only partially understood and do not always occur. In this work, we study these phenomena in the stop codon mutations c.109G>T (p.Glu37*) and c.504_505delCT; the second and third most frequent mutations in HMG-CoA lyase deficiency (MIM #246450). The deficiency affects the synthesis of ketone bodies and produces severe disorders during early childhood. We used a minigene approach, real-time quantitative PCR and the inhibition of NMD by puromycin treatment, to study the effect of stop codons on splicing (NAS) and NMD in seven patients. Surprisingly, none of the stop codons studied appears to be the direct cause of aberrant splicing. In the mutation c.109G>T, the splicing is due to the base change G>T at position 109, which is critical and cannot be explained by disruption of exonic splicing enhancer (ESE) elements, by the appearance of exonic splicing silencer (ESS) elements which were predicted by bioinformatic tools or by the stop codons. Moreover, the mutation c.504_505delCT produces two mRNA transcripts both with stop codons that generate simultaneous NMD phenomena. The effects of the mutations studied on splicing seemed to be similar in all the patients. Furthermore, we report a Spanish patient with 3-hydroxy-3-methylglutaric aciduria and a novel missense mutation: c.825C>G (p.Asn275Lys)., Diputación General de Aragón; European Social Fund; the University of Zaragoza (UZ2007-BIO-13, PIF-UZ-2009-BIO-02)

The genes HMGCS2 and HMGCL encode the two main enzymes for ketone-body synthesis, mitochondrial HMG-CoA synthase and HMG-CoA lyase. Here, we identify and describe possible splice variants of these genes in human tissues. We detected an alternative transcript of HMGCS2 carrying a deletion of exon 4, and two alternative transcripts of HMGCL with deletions of exons 5 and 6, and exons 5, 6 and 7, respectively. All splice variants maintained the reading frame. However, Western blot studies and overexpression measurements in eukaryotic or prokaryotic cell models did not reveal HL or mHS protein variants. Both genes showed a similar distribution of the inactive variants in different tissues. Surprisingly, the highest percentages were found in tissues where almost no ketone bodies are synthesized: heart, skeletal muscle and brain. Our results suggest that alternative splicing might coordinately block the two main enzymes of ketogenesis in specific human tissues, Diputación General de Aragón; University of Zaragoza UZ2007-BIO-13); Spanish Ministry of Education and Science ; Instituto de Salud Carlos III (CIBER Fisiopatologıa de la Obesidad y Nutrición); European Union (FP7-223431 ‘‘Divinocell’’ project); Fundación Ramón Areces

Cornelia de Lange syndrome (CdLS) is a dominant disorder with classic severe forms and milder atypical variants. Central to making the diagnosis is identification of diagnostic facial features. With the recognition that patients with SMC1A and SMC3 mutations have milder, atypical features, we surveyed 65 dysmorphologists using facial photographs from 32 CdLS patients with the goals of (1) Illustrating examples of milder patients with SMC1A mutations and (2) Obtaining objective data to determine which facial features were useful and misleading in making a diagnosis of CdLS. Clinicians were surveyed whether the patient had CdLS or another diagnosis, the certainty of response and the clinical features used to support each response. Using only facial photographs, an average of 24 cases (75%) were accurately diagnosed per clinician. Correct diagnoses were made in 90% of classic CdLS and 87% of non-CdLS cases, however, only 54% of mild or variant CdLS were correctly diagnosed by respondents. We confirmed that CdLS is most accurately diagnosed in childhood and the diagnosis becomes increasingly difficult with age. This survey demonstrated that emphasis is placed on the eyebrows, nasal features, prominent upper lip and micrognathia. In addition, the presence of fuller, atypical eyebrows, a prominent nasal bridge and significant prognathism with age dissuaded survey takers from arriving at a diagnosis of CdLS in individuals with mild NIPBL and SMC1A mutations. This work underscores the difficulty in diagnosing patients with mild and variant CdLS and serves to objectively classify both useful and misleading features in the diagnosis of CdLS.

Cornelia de Lange syndrome (CdLS) manifests facial dysmorphic features, growth and cognitive impairment, and limb malformations. Mutations in three genes (NIPBL, SMC1A, and SMC3) of the cohesin complex and its regulators have been found in affected patients. Here, we present clinical and molecular characterization of 30 unrelated patients with CdLS. Eleven patients had mutations in NIPBL (37%) and three patients had mutations in SMC1A (10%), giving an overall rate of mutations of 47%. Several patients shared the same mutation in NIPBL (p.R827GfsX2) but had variable phenotypes, indicating the influence of modifiers in CdLS. Patients with NIPBL mutations had a more severe phenotype than those with mutations in SMC1A or those without identified mutations. However, a high incidence of palate defects was noted in patients with SMC1A mutations. In addition, we observed a similar phenotype in both male and female patients with SMC1A mutations. Finally, we report the first patient with an SMC1A mutation and the Sandifer complex.

3-Hydroxy-3-methylglutaryl-CoA lyase (HL) deficiency is a rare autosomal recessive genetic disorder that affects ketogenesis and l-leucine catabolism, which generally appears during the first year of life. Patients with HL deficiency have a reduced capacity to synthesize ketone bodies. The disease is caused by lethal mutations in the HL gene (HMGCL). To date, up to 30 variant alleles (28 mutations and 2 SNPs) in 93 patients have been reported, with a recognizable population-specific mutational spectrum. This disorder is frequent in Saudi Arabia and the Iberian Peninsula (Portugal and Spain), where two mutations (122G>A and 109G>A) have been identified in 87% and 94% of the cases, respectively. In most countries a few patients have a high level of allelic heterogeneity. The mutations are distributed along the gene sequences, although some clustering was observed in exon 2, conforming a possible hot spot. Recently, the crystal structures of the human and two bacterial HL have been published. These experimentally obtained structures confirmed the overall architecture, previously predicted by our group and others using bioinformatic approaches, which shows the (betaalpha)8-barrel structure of the enzyme. In addition, the crystals confirmed the presence of an additional COOH domain containing important structures and residues for enzyme functionality and oligomerization processes. Here, we review all HMGCL mis-sense mutations identified to date, and their implication in enzyme structure and function is discussed. We found that genotype-phenotype correlations are difficult to establish because the evolution of the disease seems more related to the causes of hypoglycaemia (fasting or acute illness) than to a particular genotype.

HMG-CoA lyase (HL) deficiency is a rare autosomal recessive genetic disorder that affects ketogenesis and leucine catabolism. We report a new Spanish patient who bears the frequent nonsense mutation G109T (Mediterranean mutation). This mutation can produce aberrant splicing with three mRNA variants: one of the expected size, the second with deletion of exon 2, and the third with deletion of exons 2 and 3. Recently our group proposed a 3D model for human HL containing a (beta-alpha)8 (TIM) barrel structure. We have studied the effect of the deletions of exon 2 and exons 2 plus 3 on the proposed HL model. Exon 2 skipping led to the loss of beta-sheet 1, and the skipping of exons 2 and 3 caused the disappearance of alpha helix 1 and beta-sheets 1 and 2.

3-Hydroxy-3-methylglutaric aciduria is a human autosomal recessive metabolic disorder that usually appears within the first year of life. The causes of this aciduria are lethal mutations in the gene encoding for 3-hydroxy-3-methylglutaryl coenzyme A lyase (HL). HL is a mitochondrial matrix enzyme that catalyzes the last step of ketogenesis and leucine catabolism. This gene has been mapped to chromosome 1 at locus 1pter-p33 and its genomic organisation comprises 9 exons whose sizes vary between 64-678 bp. The human cDNA sequence was reported in 1993 with the first genetic study of two Acadian-French Canadian siblings. To date, 24 mutations in 36 patients have been described; most of them are single-base substitutions causing amino acid replacements and a variety of splicing defects. In the population studied two mutations appear predominant: g.122G®A (8 patients and 15 alleles) frequent in Saudi Arabia, and g.109G®T (6 patients and 12 alleles), prevalent in Spain. At least seven mutations are clustered in the second half of exon 2 affecting aminoacids E37, R41 and D42 and conforming a possible hot spot. The genotype-phenotype correlation is difficult to establish since the probands received different treatments, and the onset of an acute episode frequently depends on external factors such as fasting or acute illness.

Two novel point mutations in the 3-hydroxy-3-methylglutaryl coenzyme A lyase gene were found in a French patient with double heterozygous 3-hydroxy-3-methylglutaric aciduria. Amplification by reverse transcriptase–polymerase chain reaction of the mRNA using five different pairs of oligonucleotides produced no differences in the fragments amplified with respect to the control. Single-strand conformation polymorphism analysis showed that only one amplified fragment was different in the patient vs. control. Sequencing of the amplified fragments showed two missense point mutations, A698G and T788C, each of them mixed with the wild-type sequence. These mutations produced the changes H233R and L263P, leading to changes in the enzyme activity, which was largely abolished. The father and one brother of the proband were heterozygous for the L263P mutation and the mother and one daughter were heterozygous for the H233R mutation, which confirms the double-heterozygous character of the patient. Another sibling was free of the mutations. An enzymatic restriction analysis has been proposed to screen the occurrence of these two mutations in future patients.

3-Hydroxy-3-methylglutaric aciduria is a rare recessive monogenic disorder that affects ketogenesis and the catabolism of L-leucine. We report the biochemical and molecular characterization of a mutation in the 3-hydroxy-3-methylglutaryl coenzyme A lyase gene in four new probands, three Spanish and one Turkish, affected by 3-hydroxy-3-methylglutaric aciduria, all homozygous for the nonsense mutation Glu37Ter, which was reported by our group in two probands of Portuguese and Moroccan origin (15). In addition to the aberrant mRNAs found in the two previous probands, a novel species of mature HL mRNA was observed in the patients studied here, since a new cDNA, skipped in exons 2 and 3, was obtained from the mRNAs by reverse-transcription PCR (RT-PCR). Thus, three mRNA species were produced in aberrant splicings as a result of this nonsense mutation: (i) one of the expected size that contains the premature stop codon UAA, (ii) another with a deletion of 84 bp corresponding to the whole of exon 2, and (iii) a new species found now, with a deletion of 192 bp corresponding to skipping of the whole of exons 2 and 3, whose translation product led to the loss of seven amino acids in the leader peptide and 57 amino acids in the terminal domain of the mature enzyme. The association of a nonsense mutation with the skipping of the exon that contains it, plus the following exon, is an unusual finding not seen previously in HL deficiencies. The mutation described here shows the highest incidence (> 37%) of total HL deficiencies reported.

A novel nonsense mutation associated with the skipping of constitutive exon 2 of the 3-hydroxy-3-methylglutaryl-CoA lyase gene was found in two patients, from Portugal and Morocco, with 3-hydroxy-3-methylglutaric acidemia. By reverse transcriptase PCR and single-strand conformational polymorphism a G–T transversion was located, at nucleotide 109, of the 3-hydroxy-3-methylglutaryl-CoA lyase cDNA, within exon 2. Two mRNAs were produced as a result of this nonsense mutation: one of the expected size that contains the premature stop codon UAA, and the other with a deletion of 84 bp corresponding to the whole of exon 2. This deletion produced the loss of the last seven amino acids of the leader peptide and the first 21 amino acids of the mature protein. The nonsense mutation was found in a purine-rich GGAAG sequence, which is equal to, or similar to, others reported to be exonic splicing enhancers (ESE). We suggest that the nonsense mutation may affect a possible ESE on exon 2, which would hinder the splice site selection and facilitate an aberrant splice with the skipping of this exon. Determination by quantitative PCR shows that the ratio of mRNA with the nonsense mutation to the mRNA with the deletion is approx. 3:1.

A novel two-base deletion in the 3-hydroxy-3-methylglutaryl coenzyme A lyase (HL) gene was found in a Spanish patient with homozygous 3-hydroxy-3-methylglutaric aciduria. Amplification by RT-PCR of the mRNAs showed that the gene was transcribed into three different mRNAs. One showed the complete deletion of exons 5 and 6 located between nucleotides 348 and 561 of the HL cDNA. The second transcript showed deletion of exon 6 only, and the third contained a two-base deletion CT in exon 6, corresponding to nucleotides 504 and 505 of the HL cDNA. These aberrant mRNAs are predicted to encode three abnormal HMG-CoA lyase proteins; the first (from skipped exons 5 and 6) lacks 71 amino acids, which represents 24% of the mature protein; the second, (from the skipping of exon 6, producing a frameshift) contains only 192 amino acids, the last 26 of which are missense amino acids preceding a stop codon; the third contains only 175 amino acids, the last 7 of which are missense. Northern blot analysis showed that the HL mRNA levels of the patient were 4% of the control. PCR quantitative analysis indicated that the mRNA lacking exons 5 and 6 was the most abundant, representing 88% of the total. The other two mRNAs represented 8% and 4%, respectively. In the genomic DNA only one CT deletion was found at positions +7 and +8 at beginning of exon 6. No mutations were observed in the splice donor, splice acceptor, or pyrimidine-rich sequences of the intronic regions flanking exons 5 and 6. All three aberrant mRNAs resulted only from the deletion of nucleotides CT. We suggest that this deletion may affect the interaction between the small nuclear ribonucleoproteins (snRNPs) and exon 6, and that, as a result, the abnormal splicing of the pre-mRNA produces two different aberrant transcripts. info:eu-repo/semantics/publishedVersion

A novel point mutation in the 3-hydroxy-3methyl-glutaryl coenzyme A lyase gene was found in a Turkish patient with homozygous 3-hydroxy-3-methylglutaric acidemia. Amplification by RT-PCR of the mRNA using a six different pairs of oligonucleotides produced no differences in four of the fragments amplified with respect to the control, but generated two fragments of different size. One was representative of a deletion of 126 bp and the other of an insertion of 78 bp. These abnormal mRNAs resulted from a G–>C transversion at the nucleotide +1 of an intron, which changed the invariant GT dinucleotide of the 5' donor splice site. This was associated with the occurrence of an alternative splicing, which led to the skipping of the whole exon of 126 bp, and also with the activation of one cryptic donor splice site in the same intron. These aberrant spliced mRNAs are predicted to encode two abnormal HMG-CoA lyase proteins: the first results in a protein with an internal deletion of 42 amino acids, whose enzyme activity is largely abolished, as the catalytic site was completely removed; the second contains 17 missense amino acids that precede a stop codon. Northern blot analysis showed that the overall content of these aberrantly spliced mRNAs in proband fibroblasts was the same as that found in control fibroblasts. However, hardly any transcript was observed corresponding to the inserted mutated mRNA when it was examined by a specific probe. To quantify the relative proportion of the two mRNAs, a quantitative RT-PCR (the DNA-mimic PCR reaction) was carried out. Results show that the proportion of the inserted mRNAs with respect to the deleted mRNA is only 1.2%. The father, mother, and two brothers of the proband were heterozygous in the G–>C mutation in the +1 nucleotide of the intron considered, while the two alleles of another brother were free of the mutation. info:eu-repo/semantics/publishedVersion

Background Cornelia de Lange syndrome (CdLS) is a dominantly inherited disorder characterized by facial dysmorphism, growth and cognitive impairment, limb malformations and multiple organ involvement. Mutations in NIPBL gene account for about 60% of patients with CdLS. This gene encodes a key regulator of the Cohesin complex, which controls sister chromatid segregation during both mitosis and meiosis. Turner syndrome (TS) results from the partial or complete absence of one of the X chromosomes, usually associated with congenital lymphedema, short stature, and gonadal dysgenesis. Case presentation Here we report a four-year-old female with CdLS due to a frameshift mutation in the NIPBL gene (c.1445_1448delGAGA), who also had a tissue-specific mosaic 45,X/46,XX karyotype. The patient showed a severe form of CdLS with craniofacial dysmorphism, pre- and post-natal growth delay, cardiovascular abnormalities, hirsutism and severe psychomotor retardation with behavioural problems. She also presented with minor clinical features consistent with TS, including peripheral lymphedema and webbed neck. The NIPBL mutation was present in the two tissues analysed from different embryonic origins (peripheral blood lymphocytes and oral mucosa epithelial cells). However, the percentage of cells with monosomy X was low and variable in tissues. These findings indicate that, ontogenically, the NIPBL mutation may have appeared before the mosaic monosomy X. Conclusions The coexistence in several patients of these two rare disorders raises the issue of whether there is indeed a cause-effect association. The detailed clinical descriptions indicate predominant CdLS phenotype, although additional TS manifestations may appear in adolescence.

Mutations in the cohesin regulators NIPBL and ESCO2 are causative of the Cornelia de Lange syndrome (CdLS) and Roberts or SC phocomelia syndrome, respectively. Recently, mutations in the cohesin complex structural component SMC1A have been identified in two probands with features of CdLS. Here, we report the identification of a mutation in the gene encoding the complementary subunit of the cohesin heterodimer, SMC3, and 14 additional SMC1A mutations. All mutations are predicted to retain an open reading frame, and no truncating mutations were identified. Structural analysis of the mutant SMC3 and SMC1A proteins indicate that all are likely to produce functional cohesin complexes, but we posit that they may alter their chromosome binding dynamics. Our data indicate that SMC3 and SMC1A mutations (1) contribute to approximately 5% of cases of CdLS, (2) result in a consistently mild phenotype with absence of major structural anomalies typically associated with CdLS, and (3) in some instances, result in a phenotype that approaches that of apparently nonsyndromic mental retardation.

3‐Hydroxy‐3‐methylglutaric aciduria is a rare human autosomal recessive disorder caused by deficiency of 3‐hydroxy‐3‐methylglutaryl CoA lyase (HL). This mitochondrial enzyme catalyzes the common final step of leucine degradation and ketogenesis. Acute symptoms include vomiting, seizures and lethargy, accompanied by metabolic acidosis and hypoketotic hypoglycaemia. Such organs as the liver, brain, pancreas, and heart can also be involved. However, the pathophysiology of this disease is only partially understood. We measured mRNA levels, protein expression and enzyme activity of human HMG‐CoA lyase from liver, kidney, pancreas, testis, heart, skeletal muscle, and brain. Surprisingly, the pancreas is, after the liver, the tissue with most HL activity. However, in heart and adult brain, HL activity was not detected in the mitochondrial fraction. These findings contribute to our understanding of the enzyme function and the consequences of its deficiency and suggest the need for assessment of pancreatic damage in these patients. info:eu-repo/semantics/acceptedVersion