Assessment of Quality of Life Using the Kidslife Scale in Individuals With Cornelia de Lange Syndrome.

BACKGROUND: Cornelia de Lange syndrome (CdLS) is a rare polymalformative genetic disorder with multisystemic involvement. Despite numerous clinical and molecular studies, the specific evaluation of the quality of life (QoL) and its relationship with syndrome-specific risk factors has not been explored. METHODS: The QoL of 33 individuals diagnosed with CdLS, aged between 4 and 21 years, was assessed using the Kidslife questionnaire. Specifically, the influence of 14 risk factors on overall QoL and 8 of its domains was analyzed. RESULTS: The study revealed below-median QoL (45.3 percentile), with the most affected domains being physical well-being, personal development, and self-determination. When classifying patients based on their QoL and affected domains, variants in the NIPBL gene, clinical scores ≥11, and severe behavioral and communication issues were found to be the main risk factors. CONCLUSIONS: We emphasize the need for a comprehensive approach to CdLS that encompasses clinical, molecular, psychosocial, and emotional aspects. The "Kidslife questionnaire" proved to be a useful tool for evaluating QoL, risk factors, and the effectiveness of implemented strategies. In this study, we underscore the importance of implementing corrective measures to improve the clinical score. Furthermore, we highlight the necessity of applying specific therapies for behavioral problems after ruling out underlying causes such as pain or gastroesophageal reflux and implementing measures that facilitate communication and promote social interaction.

Cornelia de Lange Spectrum.

Cornelia de Lange syndrome (CdLS) is a rare congenital developmental disorder with multisystemic involvement. The clinical presentation is highly variable, but the classic phenotype, characterized by distinctive craniofacial features, pre- and postnatal growth retardation, extremity reduction defects, hirsutism and intellectual disability can be distinguished from the nonclassic phenotype, which is generally milder and more difficult to diagnose. In addition, the clinical features overlap with those of other neurodevelopmental disorders, so the use of consensus clinical criteria and artificial intelligence tools may be helpful in confirming the diagnosis. Pathogenic variants in NIPBL, which encodes a protein related to the cohesin complex, have been identified in more than 60% of patients, and pathogenic variants in other genes related to this complex in another 15%: SMC1A, SMC3, RAD21, and HDAC8. Technical advances in large-scale sequencing have allowed the description of additional genes (BRD4, ANKRD11, MAU2), but the lack of molecular diagnosis in 15% of individuals and the substantial clinical heterogeneity of the syndrome suggest that other genes and mechanisms may be involved. Although there is no curative treatment, there are symptomatic/palliative treatments that paediatricians should be aware of. The main medical complication in classic SCdL is gastro-esophageal reflux (GER), which should be treated early.

Heart Disease Characterization and Myocardial Strain Analysis in Patients with PACS1 Neurodevelopmental Disorder.

BACKGROUND: PACS1 neurodevelopmental disorder (PACS1-NDD) (MIM# 615009) is a rare autosomal dominant disease characterized by neurodevelopmental delay, dysmorphic facial features, and congenital malformations. Heart disease (HD) is frequently present in individuals with PACS1-NDD, but a compressive review of these anomalies and an evaluation of cardiac function in a cohort of patients are lacking. METHODS: (i) Cardiac evaluation in 11 PACS1-NDD patients was conducted using conventional echocardiography. (ii) Heart function was assessed by tissue Doppler imaging, and two-dimensional speckle tracking was performed in seven patients and matched controls. (iii) This systematic review focused on determining HD prevalence in individuals with PACS1-NDD. RESULTS: In our cohort, 7 of 11 patients presented HD. (Among them, three cases of ascending aortic dilatation (AAD) were detected and one mitral valve prolapse (MVP).) None of the patients showed echocardiographic pathological values, and the left global longitudinal strain was not significantly different between patients and controls (patients -24.26 ± 5.89% vs. controls -20.19 ± 1.75%, p = 0.3176). In the literature review, almost 42% (42/100) of individuals with PACS1-NDD reportedly experienced HD. Septal defects were the most common malformation, followed by patent ductus arteriosus. CONCLUSIONS: Our results show a high prevalence of HD in PACS1-NDD patients; in this way, AAD and MVP are reported for the first time in this syndrome. Furthermore, a detailed cardiac function evaluation in our cohort did not reveal evidence of cardiac dysfunction in individuals with PACS1-NDD. Cardiology evaluation should be included for all individuals with Schuurs-Hoeijmakers syndrome.

Selection of Novel Reference Genes by RNA-Seq and Their Evaluation for Normalising Real-Time qPCR Expression Data of Anthocyanin-Related Genes in Lettuce and Wild Relatives.

Lettuce is a popular vegetable source of bioactive compounds, like anthocyanins, powerful antioxidants present in red and semi-red varieties. Selection of reliable reference genes (RGs) for the normalization of real-time quantitative PCR (qPCR) data is crucial to obtain accurate gene expression results. Among the genes with totally unrelated biological functions, six candidate RGs (ADF2, CYB5, iPGAM, SCL13, TRXL3-3, and VHA-H) with low variation in expression according to RNA-seq analyses, were selected for future expression studies of anthocyanin-related genes in three different experiments: leaf colour comparison (green vs. red) in commercial varieties; tissue comparison (leaf vs. stem) in a wild relative; and drought stress experiment in commercial and traditional varieties, and a wild relative. Expression profiles of the candidate RGs were obtained by qPCR and their stability was assessed by four different analytical tools, geNorm, NormFinder, BestKeeper, and Delta Ct method, all integrated in RefFinder. All results considered, we recommend CYB5 to be used as RG for the leaf colour experiment and TRXL3-3 for the tissue and drought stress ones, as they were the most stable genes in each case. RNA-seq is useful to preselect novel RGs although validation by qPCR is still advisable. These results provide helpful information for gene expression studies in Lactuca spp. under the described conditions.

Endocrine Evaluation and Homeostatic Model Assessment in Patients with Cornelia de Lange Syndrome.

Objective: The aim of this study was to expand knowledge about endocrine disorders in individuals with Cornelia de Lange syndrome (CdLS), a rare developmental genetic disorder with anomalies in multiple organs and systems. Methods: Hormone levels, clinical scores, anthropometric measurements, and molecular analysis were assessed in 24 individuals with CdLS. Results: Hyperprolactinemia was the most common endocrine disorder. Three patients showed subclinical hypothyroidism. In the gonadotropic axis, mildly delayed puberty was observed, as well as genital anomalies, such as cryptorchidism. Despite short stature, levels of insulin-like growth factor 1 and insulin-like growth factor-binding protein 3 were normal, on average. Three prepubertal individuals without risk factors had higher than normal values for the homeostatic model assessment of insulin resistance (HOMA-IR) and for insulinemia, suggesting insulin resistance. Furthermore, two adults had elevated BMIs associated with HOMA-IR values over the cut-off values. Conclusion: CdLS can lead to dysregulation of the endocrine system, particularly in patients with high HOMA-IR values and insulinemia who are at risk of insulin resistance. Therefore, clinical follow-ups with hormonal assessments are proposed for individuals with CdLS.

Subclinical myocardial dysfunction is revealed by speckle tracking echocardiography in patients with Cornelia de Lange syndrome.

This study assesses a possible cardiac dysfunction in individuals with Cornelia de Lange syndrome (CdLS) without diagnosed congenital heart disease (CHD) and its association with other factors. Twenty patients and 20 controls were included in the study divided into three age-dependent groups (A: < 10 yrs, B: 10-20 yrs, C: > 20 yrs), and were evaluated using conventional echocardiography, tissue doppler imaging (TDI), two-dimensional speckle tracking and genetic and biochemical analyses. The left ventricular global longitudinal strain (GLS) was altered (< 15.9%) in 55% of patients, being pathological in the older group (A: 19.7 ± 6.6; B: -17.2 ± 4.7; C: -13.6 ± 2.9). The speckle tracking technique revealed a downward trend in the values of strain, strain rate and velocity, especially in the oldest group. Likewise, the ejection fraction (LVEF) and shortening fraction (LVFS) values, although preserved, also showed a decreased with age (p < 0.05). The analytical markers of cardiovascular risk and cardiac function showed no alterations. The molecular analyses revealed 16 individuals carrying pathogenic variants in NIPBL, two with variants in SMC1A, one with a variant in RAD21 and one with a HDAC8 variant. This is the first systematic approach that demonstrates that individuals with CdLS may present early cardiomyopathy, which can be detected by speckle tracking technique even before the appearance of clinical symptoms and the alteration of other echocardiographic or analytical parameters. For all these reasons, cardiological followup is suggested even in the absence of CHD, especially from adolescence onwards.

Case report: A novel case of parental mosaicism in SMC1A gene causes inherited Cornelia de Lange syndrome.

Ultimate advances in genetic technologies have permitted the detection of transmitted cases of congenital diseases due to parental gonadosomatic mosaicism. Regarding Cornelia de Lange syndrome (CdLS), up to date, only a few cases are known to follow this inheritance pattern. However, the high prevalence of somatic mosaicism recently reported in this syndrome (∼13%), together with the disparity observed in tissue distribution of the causal variant, suggests that its prevalence in this disorder could be underestimated. Here, we report a new case of parental gonadosomatic mosaicism in SMC1A gene that causes inherited CdLS, in which the mother of the patient carries the causative variant in very low allele frequencies in buccal swab and blood. While the affected child presents with typical CdLS phenotype, his mother does not show any clinical manifestations. As regards SMC1A, the difficulty of clinical identification of carrier females has been already recognized, as well as the gender differences observed in CdLS expressivity when the causal variant is found in this gene. Currently, the use of DNA deep-sequencing techniques is highly recommended when it comes to molecular diagnosis of patients, as well as in co-segregation studies. These enable us to uncover gonadosomatic mosaic events in asymptomatic or oligosymptomatic parents that had been overlooked so far, which might have great implications regarding genetic counseling for recurrence risk.

Molecular Basis of the Schuurs-Hoeijmakers Syndrome: What We Know about the Gene and the PACS-1 Protein and Novel Therapeutic Approaches.

The Schuurs−Hoeijmakers syndrome (SHMS) or PACS1 Neurodevelopment Disorder (PACS1-NDD) is a rare autosomal dominant disease caused by mutations in the PACS1 gene. To date, only 87 patients have been reported and, surprisingly, most of them carry the same variant (c.607C>T; p.R203W). The most relevant clinical features of the syndrome include neurodevelopment delay, seizures or a recognizable facial phenotype. Moreover, some of these characteristics overlap with other syndromes, such as the PACS2 or Wdr37 syndromes. The encoded protein phosphofurin acid cluster sorting 1 (PACS-1) is able to bind to different client proteins and direct them to their subcellular final locations. Therefore, although its main function is protein trafficking, it could perform other roles related to its client proteins. In patients with PACS1-NDD, a gain-of-function or a dominant negative mechanism for the mutated protein has been suggested. This, together with the fact that most of the patients carry the same genetic variant, makes it a good candidate for novel therapeutic approaches directed to decreasing the toxic effect of the mutated protein. Some of these strategies include the use of antisense oligonucleotides (ASOs) or targeting of its client proteins.

A Novel Intragenic Duplication in the HDAC8 Gene Underlying a Case of Cornelia de Lange Syndrome.

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.

Clinical relevance of postzygotic mosaicism in Cornelia de Lange syndrome and purifying selection of NIPBL variants in blood.

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.

Evaluating Face2Gene as a Tool to Identify Cornelia de Lange Syndrome by Facial Phenotypes.

Characteristic or classic phenotype of Cornelia de Lange syndrome (CdLS) is associated with a recognisable facial pattern. However, the heterogeneity in causal genes and the presence of overlapping syndromes have made it increasingly difficult to diagnose only by clinical features. DeepGestalt technology, and its app Face2Gene, is having a growing impact on the diagnosis and management of genetic diseases by analysing the features of affected individuals. Here, we performed a phenotypic study on a cohort of 49 individuals harbouring causative variants in known CdLS genes in order to evaluate Face2Gene utility and sensitivity in the clinical diagnosis of CdLS. Based on the profile images of patients, a diagnosis of CdLS was within the top five predicted syndromes for 97.9% of our cases and even listed as first prediction for 83.7%. The age of patients did not seem to affect the prediction accuracy, whereas our results indicate a correlation between the clinical score and affected genes. Furthermore, each gene presents a different pattern recognition that may be used to develop new neural networks with the goal of separating different genetic subtypes in CdLS. Overall, we conclude that computer-assisted image analysis based on deep learning could support the clinical diagnosis of CdLS.

More Than One HMG-CoA Lyase: The Classical Mitochondrial Enzyme Plus the Peroxisomal and the Cytosolic Ones.

There are three human enzymes with HMG-CoA lyase activity that are able to synthesize ketone bodies in different subcellular compartments. The mitochondrial HMG-CoA lyase was the first to be described, and catalyzes the cleavage of 3-hydroxy-3-methylglutaryl CoA to acetoacetate and acetyl-CoA, the common final step in ketogenesis and leucine catabolism. This protein is mainly expressed in the liver and its function is metabolic, since it produces ketone bodies as energetic fuels when glucose levels are low. Another isoform is encoded by the same gene for the mitochondrial HMG-CoA lyase (HMGCL), but it is located in peroxisomes. The last HMG-CoA lyase to be described is encoded by a different gene, HMGCLL1, and is located in the cytosolic side of the endoplasmic reticulum membrane. Some activity assays and tissue distribution of this enzyme have shown the brain and lung as key tissues for studying its function. Although the roles of the peroxisomal and cytosolic HMG-CoA lyases remain unknown, recent studies highlight the role of ketone bodies in metabolic remodeling, homeostasis, and signaling, providing new insights into the molecular and cellular function of these enzymes.

SAF-A Regulates Interphase Chromosome Structure through Oligomerization with Chromatin-Associated RNAs.

Higher eukaryotic chromosomes are organized into topologically constrained functional domains; however, the molecular mechanisms required to sustain these complex interphase chromatin structures are unknown. A stable matrix underpinning nuclear organization was hypothesized, but the idea was abandoned as more dynamic models of chromatin behavior became prevalent. Here, we report that scaffold attachment factor A (SAF-A), originally identified as a structural nuclear protein, interacts with chromatin-associated RNAs (caRNAs) via its RGG domain to regulate human interphase chromatin structures in a transcription-dependent manner. Mechanistically, this is dependent on SAF-A's AAA+ ATPase domain, which mediates cycles of protein oligomerization with caRNAs, in response to ATP binding and hydrolysis. SAF-A oligomerization decompacts large-scale chromatin structure while SAF-A loss or monomerization promotes aberrant chromosome folding and accumulation of genome damage. Our results show that SAF-A and caRNAs form a dynamic, transcriptionally responsive chromatin mesh that organizes large-scale chromosome structures and protects the genome from instability.

New case of mitochondrial HMG-CoA synthase deficiency. Functional analysis of eight mutations.

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).

Analysis of aberrant splicing and nonsense-mediated decay of the stop codon mutations c.109G>T and c.504_505delCT in 7 patients with HMG-CoA lyase deficiency.

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).

Characterization of a novel HMG-CoA lyase enzyme with a dual location in endoplasmic reticulum and cytosol.

A novel lyase activity enzyme is characterized for the first time: HMG-CoA lyase-like1 (er-cHL), which is a close homolog of mitochondrial HMG-CoA lyase (mHL). Initial data show that there are nine mature transcripts for the novel gene HMGCLL1, although none of them has all its exons. The most abundant transcript is called "variant b," and it lacks exons 2 and 3. Moreover, a three-dimensional model of the novel enzyme is proposed. Colocalization studies show a dual location of the er-cHL in the endoplasmic reticulum (ER) and cytosol, but not in mitochondria or peroxisomes. Furthermore, the dissociation experiment suggests that it is a nonendoplasmic reticulum integral membrane protein. The kinetic parameters of er-cHL indicate that it has a lower V(max) and a higher substrate affinity than mHL. Protein expression and lyase activity were found in several tissues, and were particularly strong in lung and kidney. The occurrence of er-cHL in brain is surprising, as mHL has not been found there. Although mHL activity is clearly associated with energy metabolism, the results suggest that er-cHL is more closely related to another metabolic function, mostly at the pulmonary and brain level.

Cornelia de Lange syndrome with NIPBL mutation and mosaic Turner syndrome in the same individual.

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.

Characterization of splice variants of the genes encoding human mitochondrial HMG-CoA lyase and HMG-CoA synthase, the main enzymes of the ketogenesis pathway.

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.

Differential HMG-CoA lyase expression in human tissues provides clues about 3-hydroxy-3-methylglutaric aciduria.

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.