SPEN haploinsufficiency causes a neurodevelopmental disorder overlapping proximal 1p36 deletion syndrome with an episignature of X chromosomes in females.

Deletion 1p36 (del1p36) syndrome is the most common human disorder resulting from a terminal autosomal deletion. This condition is molecularly and clinically heterogeneous. Deletions involving two non-overlapping regions, known as the distal (telomeric) and proximal (centromeric) critical regions, are sufficient to cause the majority of the recurrent clinical features, although with different facial features and dysmorphisms. SPEN encodes a transcriptional repressor commonly deleted in proximal del1p36 syndrome and is located centromeric to the proximal 1p36 critical region. Here, we used clinical data from 34 individuals with truncating variants in SPEN to define a neurodevelopmental disorder presenting with features that overlap considerably with those of proximal del1p36 syndrome. The clinical profile of this disease includes developmental delay/intellectual disability, autism spectrum disorder, anxiety, aggressive behavior, attention deficit disorder, hypotonia, brain and spine anomalies, congenital heart defects, high/narrow palate, facial dysmorphisms, and obesity/increased BMI, especially in females. SPEN also emerges as a relevant gene for del1p36 syndrome by co-expression analyses. Finally, we show that haploinsufficiency of SPEN is associated with a distinctive DNA methylation episignature of the X chromosome in affected females, providing further evidence of a specific contribution of the protein to the epigenetic control of this chromosome, and a paradigm of an X chromosome-specific episignature that classifies syndromic traits. We conclude that SPEN is required for multiple developmental processes and SPEN haploinsufficiency is a major contributor to a disorder associated with deletions centromeric to the previously established 1p36 critical regions.

Expanding the spectrum of phenotypes for MPDZ: Report of four unrelated families and review of the literature.

MPDZ, a gene with diverse functions mediating cell-cell junction interactions, receptor signaling, and binding multivalent scaffold proteins, is associated with a spectrum of clinically heterogeneous phenotypes with biallelic perturbation. Despite its clinical relevance, the mechanistic underpinnings of these variants remain elusive, underscoring the need for extensive case series and functional investigations. In this study, we conducted a systematic review of cases in the literature through two electronic databases following the PRISMA guidelines. We selected nine studies, including 18 patients, with homozygous or compound heterozygous variants in MPDZ and added five patients from four unrelated families with novel MPDZ variants. To evaluate the role of Mpdz on hearing, we analyzed available auditory electrophysiology data from a knockout murine model (Mpdzem1(IMPC)J/em1(IMPC)J) generated by the International Mouse Phenotyping Consortium. Using exome and genome sequencing, we identified three families with compound heterozygous variants, and one family with a homozygous frameshift variant. MPDZ-related disease is clinically heterogenous with hydrocephaly, vision impairment, hearing impairment and cardiovascular disease occurring most frequently. Additionally, we describe two unrelated patients with spasticity, expanding the phenotypic spectrum. Our murine analysis of the Mpdzem1(IMPC)J/em1(IMPC)J allele showed severe hearing impairment. Overall, we expand understanding of MPDZ-related phenotypes and highlight hearing impairment and spasticity among the heterogeneous phenotypes.

MicroRNA-Mediated Antiproliferative Effects of M1 Macrophage-Derived Extracellular Vesicles on Melanoma Cells.

INTRODUCTION: Research in tumor treatment has shown promising results using extracellular vesicles (EVs) derived from immune cells. EVs derived from M1 macrophages (proinflammatory), known as M1-EVs, have properties that suppress tumor growth, making them a promising treatment tool for immune susceptible tumors such as melanoma. Here, small unaltered M1-EVs (M1-sEVs) were employed in a 3D mouse melanoma model (melanospheres) to evaluate such activity. METHODS: Macrophages were polarized and EVs were isolated by ultracentrifugation. The EVs obtained were characterized based on size, with measurements performed by dynamic light scattering and electron microscopy, and the expression profiles of microRNAs were analyzed by microarray and PCR. Melanospheres were used to evaluate the cytotoxicity of M1-sEVs. Pondering a possible future transposition from the animal model to the human, human melanoma cells were transfected with a specific miRNA, and the impact on cell proliferation was evaluated. RESULTS: The isolated EVs showed a size distribution between 50-400 nm in diameter, but preeminently in a range of 70-90 nm. M1-sEVs demonstrated a remarkable ability to reduce cell proliferation and viability in the melanospheres, leading to a decrease in their volume. M1-sEVs contained unique miRNAs, including miR-29a-3p, which exhibited significant antitumor activities according to bioinformatics analysis. Validation of the antitumor effects of miR-29a-3p was obtained by a functional evaluation, i.e., by inducing miRNA overexpression in human melanoma cells (SK-MEL-28). CONCLUSION: Although further research would be advisable, the study provides evidence supporting the potential of M1-sEVs and their miRNA load as a possible targeted immune therapy for melanoma.

Early role for a Na+,K+-ATPase (ATP1A3) in brain development.

Osmotic equilibrium and membrane potential in animal cells depend on concentration gradients of sodium (Na+) and potassium (K+) ions across the plasma membrane, a function catalyzed by the Na+,K+-ATPase α-subunit. Here, we describe ATP1A3 variants encoding dysfunctional α3-subunits in children affected by polymicrogyria, a developmental malformation of the cerebral cortex characterized by abnormal folding and laminar organization. To gain cell-biological insights into the spatiotemporal dynamics of prenatal ATP1A3 expression, we built an ATP1A3 transcriptional atlas of fetal cortical development using mRNA in situ hybridization and transcriptomic profiling of ∼125,000 individual cells with single-cell RNA sequencing (Drop-seq) from 11 areas of the midgestational human neocortex. We found that fetal expression of ATP1A3 is most abundant to a subset of excitatory neurons carrying transcriptional signatures of the developing subplate, yet also maintains expression in nonneuronal cell populations. Moving forward a year in human development, we profiled ∼52,000 nuclei from four areas of an infant neocortex and show that ATP1A3 expression persists throughout early postnatal development, most predominantly in inhibitory neurons, including parvalbumin interneurons in the frontal cortex. Finally, we discovered the heteromeric Na+,K+-ATPase pump complex may form nonredundant cell-type-specific α-ß isoform combinations, including α3-ß1 in excitatory neurons and α3-ß2 in inhibitory neurons. Together, the developmental malformation phenotype of affected individuals and single-cell ATP1A3 expression patterns point to a key role for α3 in human cortex development, as well as a cell-type basis for pre- and postnatal ATP1A3-associated diseases.

Semisynthetic Sesquiterpene Lactones Generated by the Sensibility of Glaucolide B to Lewis and Brønsted-Lowry Acids and Bases: Cytotoxicity and Anti-Inflammatory Activities.

Sesquiterpene lactone (SL) subtypes including hirsutinolide and cadinanolide have a controversial genesis. Metabolites of these classes are either described as natural products or as artifacts produced via the influence of solvents, chromatographic mobile phases, and adsorbents used in phytochemical studies. Based on this divergence, and to better understand the sensibility of these metabolites, different pH conditions were used to prepare semisynthetic SLs and evaluate the anti-inflammatory and antiproliferative activities. Therefore, glaucolide B (1) was treated with various Brønsted-Lowry and Lewis acids and bases-the same approach was applied to some of its derivatives-allowing us to obtain 14 semisynthetic SL derivatives, 10 of which are hereby reported for the first time. Hirsutinolide derivatives 7a (CC50 = 5.0 µM; SI = 2.5) and 7b (CC50 = 11.2 µM; SI = 2.5) and the germacranolide derivative 8a (CC50 = 3.1 µM; SI = 3.0) revealed significant cytotoxic activity and selectivity against human melanoma SK-MEL-28 cells when compared with that against non-tumoral HUVEC cells. Additionally, compounds 7a and 7c.1 showed strongly reduced interleukin-6 (IL-6) and nitrite (NOx) release in pre-treated M1 macrophages J774A.1 when stimulated with lipopolysaccharide. Despite the fact that hirsutinolide and cadinanolide SLs may be produced via plant metabolism, this study shows that acidic and alkaline extraction and solid-phase purification processes can promote their formation.

Paralog Studies Augment Gene Discovery: DDX and DHX Genes.

Members of a paralogous gene family in which variation in one gene is known to cause disease are eight times more likely to also be associated with human disease. Recent studies have elucidated DHX30 and DDX3X as genes for which pathogenic variant alleles are involved in neurodevelopmental disorders. We hypothesized that variants in paralogous genes encoding members of the DExD/H-box RNA helicase superfamily might also underlie developmental delay and/or intellectual disability (DD and/or ID) disease phenotypes. Here we describe 15 unrelated individuals who have DD and/or ID, central nervous system (CNS) dysfunction, vertebral anomalies, and dysmorphic features and were found to have probably damaging variants in DExD/H-box RNA helicase genes. In addition, these individuals exhibit a variety of other tissue and organ system involvement including ocular, outer ear, hearing, cardiac, and kidney tissues. Five individuals with homozygous (one), compound-heterozygous (two), or de novo (two) missense variants in DHX37 were identified by exome sequencing. We identified ten total individuals with missense variants in three other DDX/DHX paralogs: DHX16 (four individuals), DDX54 (three individuals), and DHX34 (three individuals). Most identified variants are rare, predicted to be damaging, and occur at conserved amino acid residues. Taken together, these 15 individuals implicate the DExD/H-box helicases in both dominantly and recessively inherited neurodevelopmental phenotypes and highlight the potential for more than one disease mechanism underlying these disorders.

Biallelic UBE4A loss-of-function variants cause intellectual disability and global developmental delay.

PURPOSE: To identify novel genes associated with intellectual disability (ID) in four unrelated families. METHODS: Here, through exome sequencing and international collaboration, we report eight individuals from four unrelated families of diverse geographic origin with biallelic loss-of-function variants in UBE4A. RESULTS: Eight evaluated individuals presented with syndromic intellectual disability and global developmental delay. Other clinical features included hypotonia, short stature, seizures, and behavior disorder. Characteristic features were appreciated in some individuals but not all; in some cases, features became more apparent with age. We demonstrated that UBE4A loss-of-function variants reduced RNA expression and protein levels in clinical samples. Mice generated to mimic patient-specific Ube4a loss-of-function variant exhibited muscular and neurological/behavioral abnormalities, some of which are suggestive of the clinical abnormalities seen in the affected individuals. CONCLUSION: These data indicate that biallelic loss-of-function variants in UBE4A cause a novel intellectual disability syndrome, suggesting that UBE4A enzyme activity is required for normal development and neurological function.

Bariatric surgery improves clinical outcomes and adiposity biomarkers but not inflammatory cytokines SAA and MCP-1 after a six-month follow-up.

Obesity is a global health problem and is associated with a chronic low-grade inflammatory state. Surgical obesity treatment is being increasingly common due to its efficacy. From this, we evaluate the metabolic state improvement and inflammation remission in patients with obesity undergoing bariatric surgery. Methods: The clinical data and serum levels of leptin and adiponectin were assessed in patients with obesity before and one, three and six months after bariatric surgery. Also, serum amyloid A (SAA), monocyte chemoattractant protein-1 (MCP-1) levels were measured during the follow-up surgery and compared with a lean group of individuals. Results: Weight loss decreased body mass index (BMI), comorbidities percentage, drugs use and leptin levels. Adiponectin levels increased after surgery. SAA and MCP-1 showed no difference after surgery, but a trend decrease for MCP-1 and a significant decrease was observed when the patients with obesity were compared to the lean participants. Conclusion: Bariatric surgery alters metabolic status improving obesity-related comorbidities and the adiposity biomarkers leptin and adiponectin, but not inflammatory cytokines SAA and MCP-1.

In vitro cytotoxicity of co-exposure to superparamagnetic iron oxide and solid lipid nanoparticles.

Increased production and use of different types of nanoparticles (NPs) in the last decades has led to increased environmental release of these NPs with potential detrimental effects on both the environment and public health. Information is scarce in the literature on the cytotoxic effect of co-exposure to many NPs as this concern is relatively recent. Thus, in this study, we hypothesized scenarios of cell's co-exposure to two kinds of NPs, solid lipid nanoparticles (SLNs) and superparamagnetic iron oxide nanoparticles (SPIONs), to assess the potential cytotoxicity of exposure to NPs combination. Cytotoxicity of SPIONs, SLNs, and their 1:1 mixture (MIX) in six tumor and six non-tumor cell lines was investigated. The mechanisms underlining the induced cytotoxicity were studied through cell cycle analysis, detection of reactive oxygen species (ROS), and alterations in mitochondrial membrane potential (ΔΨM). Double staining with acridine orange and ethidium bromide was also used to confirm cell morphology alterations. The results showed that SPIONs induced low cytotoxicity compared to SLNs. However, the mixture of SPIONs and SLNs showed synergistic, antagonistic, and additive effects based on distinct tests such as viability assay, ROS generation, ΔΨM, and DNA damage, depending on the cell line. Apoptosis triggered by ROS and disturbances in ΔΨM are the most probable related mechanisms of action. As was postulated, there is possible cytotoxic interaction between the two kinds of NPs.

The fructose-1,6-bisphosphatase deficiency and the p.(Lys204ArgfsTer72) variant.

Fructose-1,6-bisphosphatase (FBPase) deficiency is a rare inborn error of fructose metabolism caused by pathogenic variants in the FBP1 gene. As gluconeogenesis is affected, catabolic episodes can induce ketotic hypoglycemia in patients. FBP1 analysis is the most commonly used approach for the diagnosis of this disorder. Herein, a Brazilian patient is reported. The proband, a girl born to a consanguineous couple, presented with severe hypoglycemia crisis in the neonatal period. At the age 17 months, presented a new crisis accompanied by metabolic acidosis associated with a feverish episode. Genetic analysis was performed by next-generation sequencing (NGS), identifying the NM_000507.3:c.611_614del variant in homozygosis in the FBP1 gene. In silico analysis and 3D modeling were performed, suggesting that this variant is associated with a loss of sites for substrate and Mg2+ binding and for posttranslational modifications of FBPase. The c.611_614del variant is located in a repetitive region of the FBP1 gene that appears to be a hotspot for mutational events. This frameshift creates a premature termination codon in the last coding exon which escapes the nonsense-mediated decay mechanism, according to in silico analysis. This variant results in an intrinsically disordered protein with loss of substrate recognition and post-translational modification sites.

Loss of TNR causes a nonprogressive neurodevelopmental disorder with spasticity and transient opisthotonus.

PURPOSE: TNR, encoding Tenascin-R, is an extracellular matrix glycoprotein involved in neurite outgrowth and neural cell adhesion, proliferation and migration, axonal guidance, myelination, and synaptic plasticity. Tenascin-R is exclusively expressed in the central nervous system with highest expression after birth. The protein is crucial in the formation of perineuronal nets that ensheath interneurons. However, the role of Tenascin-R in human pathology is largely unknown. We aimed to establish TNR as a human disease gene and unravel the associated clinical spectrum. METHODS: Exome sequencing and an online matchmaking tool were used to identify patients with biallelic variants in TNR. RESULTS: We identified 13 individuals from 8 unrelated families with biallelic variants in TNR sharing a phenotype consisting of spastic para- or tetraparesis, axial muscular hypotonia, developmental delay, and transient opisthotonus. Four homozygous loss-of-function and four different missense variants were identified. CONCLUSION: We establish TNR as a disease gene for an autosomal recessive nonprogressive neurodevelopmental disorder with spasticity and transient opisthotonus and highlight the role of central nervous system extracellular matrix proteins in the pathogenicity of spastic disorders.

Mechanism of action of camphoryl-benzene sulfonamide derivative on glucose uptake in adipose tissue.

The aim of the present study was to investigate the mechanism of action of a sulfonamide derivative on glucose uptake in adipose tissue, as well as to characterize the effects of this compound on intestinal disaccharidases and advanced glycation end-products (AGEs) formation. Camphoryl-benzene sulfonamide (CS) was able to stimulate glucose uptake in isolated adipocytes, adipose tissue, and in soleus muscle. The stimulatory effect of the compound (10 µM) on glucose uptake on adipose tissue was blocked by diazoxide, wortmannin, U73122, colchicine, and N-ethylmaleimide. On the other hand, the effects of CS were not blocked by glibenclamide, an inhibitor of the K+ -ATP channel, or even by the inhibitor of protein p38 MAPK, SB 203580. In vivo, this compound reduced intestinal disaccharidase activity, while, in vitro, CS reduced the formation of AGEs at 7, 14, and 28 days of incubation. The stimulatory effect of CS on glucose uptake requires the activation of the K+ -ATP channel, translocation, and fusion of GLUT4 vesicles to the plasma membrane on adipocytes for glucose homeostasis. In addition, the inhibition of disaccharidase activity contributes to the glucose homeostasis in a short-term as well as the remarkable reduction in AGE formation indicates that the CS may prevent of complications of late diabetes.

Contact lens-related polymicrobial keratitis: Acanthamoeba spp. genotype T4 and Candida albicans.

A 31-year-old female daily user of contact lenses sought medical attention, reporting blurred vision and irritation of the left eye. Slit-lamp examination revealed hyperemia and an irregular corneal epithelium surface, and empirical treatment was started. A corneal scrape was obtained and examined for the presence of fungi, bacteria, and Acanthamoeba spp. The results of the microbial culture revealed growth of Acanthamoeba spp. and Candida albicans. The Acanthamoeba isolate was characterized by cyst morphology as belonging to group II according to Pussard and Pons. Sequencing of the diagnostic fragment 3 (DF3) region located on the 18S ribosomal DNA identified the isolate as genotype T4. The patient was treated with chlorhexidine 0.02% and polyhexamethylene biguanide (PHMB) 0.02% drops for 5 months until the infection resolved. Lately, rare cases of polymicrobial keratitis associated with Acanthamoeba and Candida albicans have been reported. Cases of co-infection are more difficult to treat, since the specific treatment depends on precise identification of the agents involved.

UPLC-ESI-QTOF-MS2 characterisation of Cola nitida resin fractions with inhibitory effects on NO and TNF-α released by LPS-activated J774 macrophage and on Trypanosoma cruzi and Leishmania amazonensis.

INTRODUCTION: The resin of Cola nitida is used in western Cameroon as incense for spiritual protection and during ritual ceremonies. This plant secretion has never been investigated although previous chemical and biological studies on other resins have drawn many attentions. OBJECTIVE: The resin fractions which revealed inhibitory effect on nitric oxide (NO) and tumour necrosis factor alpha (TNF-α) released by lipopolysaccharide (LPS)-activated J774 macrophage as well as on intracellular forms of Leishmania amazonensis and Trypanosoma cruzi amastigote were chemically characterised. Moreover, their antiparasitic activities were compared to those of semi-synthetic triterpenes. METHODOLOGY: The anti-inflammatory activity was evaluated by measuring the nitrite production and the TNF-α concentration in the supernatants of LPS-activated macrophages by antigen capture enzyme-linked immunosorbent assay. Moreover, the antiparasitic assay was performed by infecting the host cells (THP-1) in a ratio parasite/cell 10:1 (L. amazonensis) and 2:1 (T. cruzi) and then exposed to the samples. The resin was separated in vacuo by liquid chromatography because of its sticky behaviour and the chemical profiles of the obtained fractions (F1-F4) were established by dereplication based on UPLC-ESI-MS2 data while semi-synthetic triterpenes were prepared from α-amyrin by oxidation reactions. RESULTS: Fractions F1-F4 inhibited NO and TNF-α almost similarly. However, only F1, F3 and F4 showed promising antiparasitic activities while F2 was moderately active against both parasites. Hence, F1-F4 were exclusively composed of pentacyclic triterpenes bearing oleanane and ursane skeletons. Semi-synthetic compounds revealed no to moderate antiparasitic activity compared to the fractions. CONCLUSION: Although it will be difficult to prove the interaction resin-spirit, interesting bioactivities were found in the resin fractions.

Expanding the Molecular and Clinical Phenotype of SSR4-CDG.

Congenital disorders of glycosylation (CDG) are a group of mostly autosomal recessive disorders primarily characterized by neurological abnormalities. Recently, we described a single CDG patient with a de novo mutation in the X-linked gene, Signal Sequence Receptor 4 (SSR4). We performed whole-exome sequencing to identify causal variants in several affected individuals who had either an undifferentiated neurological disorder or unsolved CDG of unknown etiology based on abnormal transferrin glycosylation. We now report eight affected males with either de novo (4) or inherited (4) loss of function mutations in SSR4. Western blot analysis revealed that the mutations caused a complete loss of SSR4 protein. In nearly all cases, the abnormal glycosylation of serum transferrin was only slightly above the accepted normal cutoff range.

Clinical Features in Patients With 22q11.2 Deletion Syndrome Ascertained by Palatal Abnormalities.

OBJECTIVES: The aim of this study was to describe clinical features in subjects with palatal abnormalities and to assess the distribution of these features among those with and without 22q11.2 deletion. DESIGN: Descriptive cohort. PATIENTS: One hundred patients with palatal abnormalities and suspicion of 22q11.2 DS were included. METHODS: All patients were evaluated by a clinical geneticist, who completed a standardized clinical protocol. The 22q11.2 deletion screening was performed with fluorescence in situ hybridization using the TUPLE1 probe and multiplex ligation-dependent probe amplification using the P250-A1 kit. RESULTS: The 22q11.2 deletion was detected in 35 patients, in whom the most frequent clinical features were congenital heart disease (15/30 - 50%), developmental delay (19/35 - 54%), speech delay (20/35 - 57%), learning disabilities (27/35 - 77%), immunologic alterations (18/29 - 62%). In addition, the most common facial dysmorphisms in this group were long face (27/35 - 77%), typical nose (24/35 - 69%), and hooded eyelids (19/35 - 54%). Comparing features in patients with or without the deletion revealed significant differences (positively correlated with the deletion) for speech delay, learning disabilities, conductive hearing loss, number of dysmorphisms, long face, and hooded eyelids. Cleft lip and palate was negatively correlated with the deletion. CONCLUSIONS: The presence of speech delay, learning disabilities, conductive hearing loss, long face, and hooded eyelids should reinforce the suspicion of 22q11.2 DS in patients with palatal abnormalities and would help professionals direct clinical follow-up of these patients.

Antitumoral activity of a trichloromethyl pyrimidine analogue: molecular cross-talk between intrinsic and extrinsic apoptosis.

Acute lymphoblastic leukemia (ALL) is a malignant disorder caused by the proliferation of lymphoid progenitor cells and is the most common cancer in children. Cytotoxic nucleoside analogues are important chemotherapeutic agents, which are used in many cancers, including leukemias. In this study, we investigated the effects of the synthetic nucleoside analogue 1-(5,5,5-trichloro-2-methoxy-4-oxopenten-2-yl)-4-trichloromethyl-pyrimidin-2(1H)-one, named compound 3 or C3, on leukemia cell lines. The compound stimulated cell death by apoptosis, evidenced by DNA fragmentation, phosphatidylserine externalization, and caspase-3 activation. Compound 3 seemed to trigger several cell death pathways. The mitochondrial pathway was evidenced through a disturbance of mitochondrial membrane potential, strong cytochrome c liberation, decrease of antiapoptotic Bcl-2 protein expression, and caspase-9 activation. The C3 also induced caspase-8 and -12 activation, an increase in the intracellular calcium level, and an overproduction of reactive oxygen species. Increased caspase 8 activity suggests that the extrinsic pathway was activated and that the ROS production and enzyme activity alteration (glutathione S-transferase, glutathione peroxidase, catalase, and glutathione reductase) might be related to oxidative stress. Finally, the increase in calcium release, CHOP expression, and caspase-12 activity might characterize endoplasmic reticulum stress. Compound 3 was likewise cytotoxic to leukemic and melanoma human cell lines. Taken together, the results contribute to further understanding the new pyrimidine analogue as a potential chemotherapeutic drug or lead molecule.

GLP1R (glucagon-like-peptide-1 incretin receptor), diabetes and obesity phenotypes: An in silico approach revealed new pathogenic variants.

OBJECTIVE: Glucagon-like peptide-1 receptor belongs to the B family of G protein-coupled receptors, serving as a binding protein in membranes and is widely expressed in human tissues. Upon stimulation by its agonist, the glucagon-like peptide-1, the receptor plays a role in glucose metabolism, enhancing insulin secretion, and regulating appetite in the hypothalamus. Mutations in the glucagon-like peptide-1 receptor gene can lead to physiological changes that may explain phenotypic variations in individuals with obesity and diabetes. Therefore, this study aimed to evaluate missense variants of the glucagon-like peptide-1 receptor gene. METHODS: Data mining was performed on the single nucleotide polymorphism database, retrieving a total of 16,399 variants. Among them, 356 were missense. These 356 variants were analyzed using the PolyPhen-2 and filtered based on allele frequency, resulting in 6 pathogenic variants. RESULTS: D344E, A239T, R310Q, R227H, R421P, and R176G were analyzed using four different prediction tools. The D344E and A239T resulted in larger amino acid residues compared to their wild-type counterparts. The D344E showed a slightly destabilized structure, while A239T affected the transmembrane helices. Conversely, the R310Q, R227H, R421P, and R176G resulted in smaller amino acid residues than the wild-type, leading to a loss of positive charge and increased hydrophobicity. Particularly, the R421P, due to the presence of proline, significantly destabilized the α-helix structure and caused severe damage to the receptor. CONCLUSION: Elucidating the glucagon-like peptide-1 receptor variants and their potentially detrimental effects on receptor functionality can contribute to an understanding of metabolic diseases and the response to available pharmacological treatments.

Menke-Hennekam syndrome; delineation of domain-specific subtypes with distinct clinical and DNA methylation profiles.

CREB-binding protein (CBP, encoded by CREBBP) and its paralog E1A-associated protein (p300, encoded by EP300) are involved in histone acetylation and transcriptional regulation. Variants that produce a null allele or disrupt the catalytic domain of either protein cause Rubinstein-Taybi syndrome (RSTS), while pathogenic missense and in-frame indel variants in parts of exons 30 and 31 cause phenotypes recently described as Menke-Hennekam syndrome (MKHK). To distinguish MKHK subtypes and define their characteristics, molecular and extended clinical data on 82 individuals (54 unpublished) with variants affecting CBP (n = 71) or p300 (n = 11) (NP_004371.2 residues 1,705-1,875 and NP_001420.2 residues 1,668-1,833, respectively) were summarized. Additionally, genome-wide DNA methylation profiles were assessed in DNA extracted from whole peripheral blood from 54 individuals. Most variants clustered closely around the zinc-binding residues of two zinc-finger domains (ZZ and TAZ2) and within the first α helix of the fourth intrinsically disordered linker (ID4) of CBP/p300. Domain-specific methylation profiles were discerned for the ZZ domain in CBP/p300 (found in nine out of 10 tested individuals) and TAZ2 domain in CBP (in 14 out of 20), while a domain-specific diagnostic episignature was refined for the ID4 domain in CBP/p300 (in 21 out of 21). Phenotypes including intellectual disability of varying degree and distinct physical features were defined for each of the regions. These findings demonstrate existence of at least three MKHK subtypes, which are domain specific (MKHK-ZZ, MKHK-TAZ2, and MKHK-ID4) rather than gene specific (CREBBP/EP300). DNA methylation episignatures enable stratification of molecular pathophysiologic entities within a gene or across a family of paralogous genes.