Screening for Rare Mitochondrial Genome Variants Reveals a Potentially Novel Association between MT-CO1 and MT-TL2 Genes and Diabetes Phenotype.

Variations in several nuclear genes predisposing humans to the development of MODY diabetes have been very well characterized by modern genetic diagnostics. However, recent reports indicate that variants in the mtDNA genome may also be associated with the diabetic phenotype. As relatively little research has addressed the entire mitochondrial genome in this regard, the aim of the present study is to evaluate the genetic variations present in mtDNA among individuals susceptible to MODY diabetes. In total, 193 patients with a MODY phenotype were tested with a custom panel with mtDNA enrichment. Heteroplasmic variants were selected for further analysis via further sequencing based on long-range PCR to evaluate the potential contribution of frequent NUMTs (acronym for nuclear mitochondrial DNA) insertions. Twelve extremely rare variants with a potential damaging character were selected, three of which were likely to be the result of NUMTs from the nuclear genome. The variant m.3243A>G in MT-TL1 was responsible for 3.5% of MODY cases in our study group. In addition, a novel, rare, and possibly pathogenic leucine variant m.12278T>C was found in MT-TL2. Our findings also found the MT-CO1 gene to be over-represented in the study group, with a clear phenotype-genotype correlation observed in one family. Our data suggest that heteroplasmic variants in MT-COI and MT-TL2 genes may play a role in the pathophysiology of glucose metabolism in humans.

SERS Signature of SARS-CoV-2 in Saliva and Nasopharyngeal Swabs: Towards Perspective COVID-19 Point-of-Care Diagnostics.

In this study, the intrinsic surface-enhanced Raman spectroscopy (SERS)-based approach coupled with chemometric analysis was adopted to establish the biochemical fingerprint of SARS-CoV-2 infected human fluids: saliva and nasopharyngeal swabs. The numerical methods, partial least squares discriminant analysis (PLS-DA) and support vector machine classification (SVMC), facilitated the spectroscopic identification of the viral-specific molecules, molecular changes, and distinct physiological signatures of pathetically altered fluids. Next, we developed the reliable classification model for fast identification and differentiation of negative CoV(-) and positive CoV(+) groups. The PLS-DA calibration model was described by a great statistical value-RMSEC and RMSECV below 0.3 and R2cal at the level of ~0.7 for both type of body fluids. The calculated diagnostic parameters for SVMC and PLS-DA at the stage of preparation of calibration model and classification of external samples simulating real diagnostic conditions evinced high accuracy, sensitivity, and specificity for saliva specimens. Here, we outlined the significant role of neopterin as the biomarker in the prediction of COVID-19 infection from nasopharyngeal swab. We also observed the increased content of nucleic acids of DNA/RNA and proteins such as ferritin as well as specific immunoglobulins. The developed SERS for SARS-CoV-2 approach allows: (i) fast, simple and non-invasive collection of analyzed specimens; (ii) fast response with the time of analysis below 15 min, and (iii) sensitive and reliable SERS-based screening of COVID-19 disease.

Elevated level of lysophosphatidic acid among patients with HNF1B mutations and its role in RCAD syndrome: a multiomic study.

INTRODUCTION: Patients with hepatocyte nuclear factor-1 beta (HNF1B) mutations present a variable phenotype with two main symptoms: maturity onset diabetes of the young (MODY) and polycystic kidney disease (PKD). OBJECTIVES: Identification of serum metabolites specific for HNF1Bmut and evaluation of their role in disease pathogenesis. METHODS: We recruited patients with HNF1Bmut (N = 10), HNF1Amut (N = 10), PKD: non-dialyzed and dialyzed (N = 8 and N = 13); and healthy controls (N = 12). Serum fingerprinting was performed by LC-QTOF-MS. Selected metabolite was validated by ELISA (enzyme-linked immunosorbent assay) measurements and then biologically connected with HNF1B by in silico analysis. HepG2 were stimulated with lysophosphatidic acid (LPA) and HNF1B gene was knocked down (kd) by small interfering RNA. Transcriptomic analysis with microarrays and western blot measurements were performed. RESULTS: Serum levels of six metabolites including: arachidonic acid, hydroxyeicosatetraenoic acid, linoleamide and three LPA (18:1, 18:2 and 20:4), had AUC (the area under the curve) > 0.9 (HNF1Bmut vs comparative groups). The increased level of LPA was confirmed by ELISA measurements. In HepG2HNF1Bkd cells LPA stimulation lead to downregulation of many pathways associated with cell cycle, lipid metabolism, and upregulation of steroid hormone metabolism and Wnt signaling. Also, increased intracellular protein level of autotaxin was detected in the cells. GSK-3alpha/beta protein level and its phosphorylated ratio were differentially affected by LPA stimulation in HNF1Bkd and control cells. CONCLUSIONS: LPA is elevated in sera of patients with HNF1Bmut. LPA contributes to the pathogenesis of HNF1B-MODY by affecting Wnt/GSK-3 signaling.

Case report: Neonatal diabetes mellitus with congenital hypothyroidism as a result of biallelic heterozygous mutations in GLIS3 gene.

Neonatal diabetes mellitus with congenital hypothyroidism (NDH) syndrome (MIM# 610199) is a rare disease caused by autosomal recessive mutations in the GLIS3 gene. GLIS3 is an important transcription factor that might acts as both a repressor and activator of transcription. To date, 22 cases of NDH syndrome from 16 families and 11 countries have been described. Herein, we report a child who developed diabetes during the first week of age. Additionally, she suffered from congenital hypothyroidism, cardiac abnormalities, and polycystic kidney disease. Genetic analysis revealed that patient is a carrier of two novel heterozygous mutations, p.Pro444fsdelG (c.1330delC) and p.His647Arg (c.1940A > G) in the GLIS3 gene. Each was inherited from clinically healthy father and mother, respectively. Bioinformatic tools (SIFT, PolyPhen2, PROVEAN and SWISS-MODEL) declared that the p.His647Arg (c.1940A > G) variant has strong detrimental effect and disturbs Kruppel-like zinc finger domain. All but one so far described cases of NDH syndrome have been caused by homozygous of GLIS3, making the described case the second case of pathogenic, compound heterozygosity of GLIS3 worldwide posing substantial clinical novelty and detailing an interesting interplay between the observed variants and GLIS3 expression, which seems to be autoregulated. Hence, the damaging missense mutation may further reduce the expression of any remaining functional alleles. This case report expands our understanding of the clinical phenotype, treatment approaches, and outcome of infants with GLIS3 mutations and indicates the need for further research to deepen our understanding of the role of GLIS3.

There and back again: a dendrimer's tale.

The development of molecular nanostructures with well-defined particle size and shape is of eminent interest in biomedicine. Among many studied nanostructures, dendrimers represent the group of those most thoroughly characterized ones. Due to their unique structure and properties, dendrimers are very attractive for medical and pharmaceutical applications. Owing to the controllable cavities inside the dendrimer, guest molecules may be encapsulated, and highly reactive terminal groups are susceptible to further modifications, e.g., to facilitate target delivery. To understand the potential of these nanoparticles and to predict and avoid any adverse cellular reactions, it is necessary to know the mechanisms responsible for an efficient dendrimer uptake and the destination of their intracellular journey. In this article, we summarize the results of studies describing the dendrimer uptake, traffic, and efflux mechanisms depending on features of specific nanoparticles and cell types. We also present mechanisms of dendrimers responsible for toxicity and alteration in signal transduction pathways at the cellular level.

Neurotoxicity of poly(propylene imine) glycodendrimers.

Published results of studies on poly(propylene imine) (PPI) dendrimers indicate their potential use in the treatment of brain cancer or neurodegenerative diseases due to their ability to cross the blood-brain barrier. However, depending on dose, neurotoxicity may occur. Here, we discuss the impact of maltotriose modified PPI dendrimers on rat's nervous system. Wistar rats were treated intravenously for 14 consecutive days with densely (dense-shell; DS) and partly (open-shell; OS) modified PPI dendrimers at doses established as safe in the previous experiment following a single DS or OS administration. The examination included an estimation of the motility and the clinical symptoms of the respiratory, nervous, and cardiovascular systems. Both DS and OS glycodendrimers (GDs) induced adverse effects at the doses tested. Multiple administrations of PPI-OS had a detrimental influence on rats' survival. These findings suggest that the dendrimers adversely influence the nervous system and their toxic effects accumulate over time. In PPI-DS treated animals, the harmful effects were less severe but still present. However, with each treatment day, the clinical symptoms in both groups were less severe as if the animals developed tolerance to GDs. We hypothesize that the neurotoxicity of tested dendrimers is related to nanoparticles-induced autophagy.

Multiomic analysis on human cell model of wolfram syndrome reveals changes in mitochondrial morphology and function.

BACKGROUND: Wolfram syndrome (WFS) is a rare autosomal recessive syndrome in which diabetes mellitus and neurodegenerative disorders occur as a result of Wolframin deficiency and increased ER stress. In addition, WFS1 deficiency leads to calcium homeostasis disturbances and can change mitochondrial dynamics. The aim of this study was to evaluate protein levels and changes in gene transcription on human WFS cell model under experimental ER stress. METHODS: We performed transcriptomic and proteomic analysis on WFS human cell model-skin fibroblasts reprogrammed into induced pluripotent stem (iPS) cells and then into neural stem cells (NSC) with subsequent ER stress induction using tunicamycin (TM). Results were cross-referenced with publicly available RNA sequencing data in hippocampi and hypothalami of mice with WFS1 deficiency. RESULTS: Proteomic analysis identified specific signal pathways that differ in NSC WFS cells from healthy ones. Next, detailed analysis of the proteins involved in the mitochondrial function showed the down-regulation of subunits of the respiratory chain complexes in NSC WFS cells, as well as the up-regulation of proteins involved in Krebs cycle and glycolysis when compared to the control cells. Based on pathway enrichment analysis we concluded that in samples from mice hippocampi the mitochondrial protein import machinery and OXPHOS were significantly down-regulated. CONCLUSIONS: Our results show the functional and morphological secondary mitochondrial damage in patients with WFS. Video Abstract.

CAR-NK Cells in the Treatment of Solid Tumors.

CAR-T (chimeric antigen receptor T) cells have emerged as a milestone in the treatment of patients with refractory B-cell neoplasms. However, despite having unprecedented efficacy against hematological malignancies, the treatment is far from flawless. Its greatest drawbacks arise from a challenging and expensive production process, strict patient eligibility criteria and serious toxicity profile. One possible solution, supported by robust research, is the replacement of T lymphocytes with NK cells for CAR expression. NK cells seem to be an attractive vehicle for CAR expression as they can be derived from multiple sources and safely infused regardless of donor-patient matching, which greatly reduces the cost of the treatment. CAR-NK cells are known to be effective against hematological malignancies, and a growing number of preclinical findings indicate that they have activity against non-hematological neoplasms. Here, we present a thorough overview of the current state of knowledge regarding the use of CAR-NK cells in treating various solid tumors.

Genetic tests based on the RT-PCR reaction in the diagnostics of SARS-CoV-2 infection.

INTRODUCTION: Since the SARS-CoV-2 emergence in 2019/2020, at least 158 million infections with this pathogen have been recorded, of which 3.29 million infected people have died. Due to the non-specific symptoms of SARS-CoV-2 infection, laboratory tests based on RT-PCR (reverse transcription and polymerase chain reaction) are mainly used in the diagnosis of COVID-19 disease. AIM: The aim of this study is to compare the molecular tests available on the Polish market for the diagnosis of SARS-CoV2 infection. RESULTS: Based on the data provided by the manufacturers and the performed laboratory analyses, we have shown that the available diagnostic kits differ mainly in the sensitivity and duration of the reaction. CONCLUSION: Due to the ongoing COVID-19 pandemic, the indicated parameters are key to effective control of the spread of SARS-CoV2, and therefore should be mainly taken into account when choosing and purchasing by diagnostic centres.

Maltotriose-modified poly(propylene imine) Glycodendrimers as a potential novel platform in the treatment of chronic lymphocytic Leukemia. A proof-of-concept pilot study in the animal model of CLL.

Cancer nanotherapeutics have shown promise in resolving some of the limitations of conventional drug delivery systems such as nonspecific biodistribution and targeting, lack of water solubility, and low therapeutic indices, Among the various nanoparticles that are available, dendrimers, highly branched macromolecules with a specific size and shape, are one of the most promising ones. In this preliminary study, we tested the anti-tumor activity of maltotriose-modified fourth-generation poly(propylene imine) glycodendrimers (PPI-G4-M3) in vivo in the subcutaneous MEC-1 xenograft model of human chronic lymphocytic leukemia (CLL) in NOD scid gamma mice. Fludarabine was used for model validation and as a positive treatment control. The anti-tumor response was calculated as tumor volume, tumor control ratio, and tumor growth inhibition. The study showed that PPI-G4-M3 inhibited subcutaneous tumor growth more efficiently than fludarabine. The anti-tumor response was dose-dependent. Cationic PPI-G4-M3 showed the highest anti-tumor activity but also higher toxicity than the neutral dendrimers and fludarabine. These first promising results warrant further studies in the optimization of dendrimers charge, dose, route and schedule of administration to combat CLL.

A cross-sectional study of patients referred for HNF1B-MODY genetic testing due to cystic kidneys and diabetes.

BACKGROUND/OBJECTIVES: Patients referred for HNF1B testing present very heterogeneous phenotypes. Despite suggestive characteristics, many do not harbor mutations in HNF1B. Our objective was to evaluate the clinical characteristics of probands referred for HNF1B genetic testing through a nationwide monogenic diabetes screening program. METHODS: Probands tested for HNF1B mutations in the 2005-2018 period (N = 50) were identified in the Polish Monogenic Diabetes Registry, which prospectively recruits primarily pediatric patients and their families on a nationwide scale. Variants that had been reported pathogenic were reassessed using criteria of the American College of Medical Genetics and Genomics (ACMG). A structured medical interview was performed with all available individuals, their parents, and/or their physicians. For each patient, HNF1B score was calculated based on available clinical information. RESULTS: The study group numbered 36 unrelated probands (28% lost to follow-up): 14 with pathogenic or likely-pathogenic variants in HNF1B, one with a variant of uncertain significance, and 21 negative for HNF1B mutations. Presence of cystic kidneys (OR = 9.17, 95% CI:1.87-44.92), pancreatic abnormalities (OR = 15, 95% CI:1.55-145.23), elevated liver enzymes (OR = 15, 95% CI:1.55-145.23) best discriminated HNF1B-positive cases from the negative ones. Presence of impaired glucose tolerance coupled with kidney disease in the proband and one parent was also highly predictive for HNF1B mutations (OR = 11.11, 95% CI:1.13-109.36). HNF1B-score with recommended cutoff distinguished patients with and without HNF1B findings with 100% sensitivity and 47.6% specificity. Addition of four clinical variables to select patients based on HNF1B score improved specificity to 71.4% (95% CI:47.8%-88.7%) while retaining 100% sensitivity. CONCLUSIONS: Detailed medical interview may enable more accurate patient selection for targeted genetic testing.

Identification of placental genes linked to selective intrauterine growth restriction (IUGR) in dichorionic twin pregnancies: gene expression profiling study.

A linkage of dichorionic (DC) twin pregnancies with selective intrauterine growth restriction (IUGR) to alterations in placental gene expression is unclear. The aim of the study was to identify placental genes related to hypoxia, adipogenesis and human growth which may contribute to IUGR development. The study group (IUGR/AGA) comprised dichorionic (DC) twin pregnancies, where the weight of the twins differed by > 15%; in addition, one twin was small for gestational age (< 10th percentile-SGA) (IUGR) while the other was appropriate for gestational age (> 10th percentile-AGA). In the control group (AGA/AGA), both fetuses were AGA and their weights differed by < 15%. In the first step (selection), placental expression of 260 genes was analysed by commercial PCR profiler array or qPCR primer assay between six pairs of IUGR/AGA twins. In the second stage (verification), the expression of 20 genes with fold change (FC) > 1.5 selected from the first stage was investigated for 75 DC pregnancies: 23 IUGR/AGA vs. 52 AGA/AGA. The expression of Angiopoetin 2, Leptin and Kruppel-like factor 4 was significantly higher, and Glis Family Zinc Finger 3 was lower, in placentas of SGA fetuses (FC = 3.3; 4.4; 1.6; and - 1.8, respectively; p < 0.05). The dysregulation of gene expression related to angiogenesis and growth factors in placentas of twins born from IUGR/AGA pregnancies suggest that these alternations might represent biological fetal adaptation to the uteral condition. Moreover, DC twin pregnancies may be a good model to identify the differences in placental gene expression between SGA and AGA fetuses.

Optical coherence tomography and magnetic resonance imaging visual pathway evaluation in Wolfram syndrome.

AIM: The aim of this study was to assess parameters of retinal morphology by using high-definition optical coherence tomography (OCT) in patients with Wolfram syndrome (WFS) and their relation to optic tract atrophy in magnetic resonance imaging (MRI). METHOD: High-definition OCT and MRI parameters were evaluated in 12 patients with WFS (three males, nine females; median age at examination 12y 8mo, range 10y 2mo-15y 11mo) and referred to 30 individuals with type 1 diabetes (T1D) (12 males, 18 females; median age at examination 20y 5mo, range 16y 8mo-21y 4mo) and 33 typically developing comparison participants (10 males, 23 females; median age at examination 20y 7mo, range 13y-22y 4mo). RESULTS: Total thickness and quadrant thickness of the retinal nerve fibre layer (RNFL), macular full-thickness parameters and macular ganglion cell layer/inner plexiform layer, intraorbital and intracranial thickness of the optical nerve, as well as the optic chiasm and visual tracts were significantly reduced in patients with WFS compared with those having T1D and the typically developing comparison participants. Optic chiasm thickness correlated negatively in patients with WFS with both age (r=-0.79; p=0.002) and duration of diabetes (r=-0.62; p=0.032). Thickness of the intraorbital parts of the optic nerves in patients with WFS correlated positively with thickness of the superior RNFL (r=0.73; p=0.006). INTERPRETATION: High-definition OCT in combination with MRI could become an important tool for evaluating the effectiveness of therapeutic trials in patients with WFS. WHAT THIS PAPER ADDS: Provides evidence of significant reduction of retinal parameters and optic nerves in patients with Wolfram syndrome (WFS). Shows correlations between magnetic resonance imaging parameters and retinal morphology parameters in patients with WFS.

Affecting NF-κB cell signaling pathway in chronic lymphocytic leukemia by dendrimers-based nanoparticles.

The complex genetic diversity of chronic lymphocytic leukemia (CLL) makes it difficult to determine the effective and durable therapy beneficial to patients. During the several past years' significant insights in the biology of the disease and its treatment have been made, allowing for the identification of promising novel therapeutic agents. The investigation of signaling pathways to understand the biological character of CLL together with the development of molecular profiling is key in personalized approach in therapy for this disease. As it was already proven, maltotriose (M3) modified fourth generation poly(propylene imine) dendrimers (PPI-G4) modulate BCR, TRAIL and WNT signaling pathway gene expression in CLL cells and strongly influence their survival by inducing apoptosis and inhibiting proliferation. The aim of this study was to evaluate the influence of PPI-G4-M3 dendrimers on NFκB pathway gene expression in CLL (MEC-1) cells with 60 K microarray, as it is one of the major factors in the pathogenesis of B-cell neoplasms. The findings were compared with those obtained with Fludarabine (FA) and the results indicate that PPI-G4-M3 dendrimers affect the expression of the examined genes and exert comparable effect on the CLL cells to FA. Dendrimers are one of the most potent groups of nanometer-sized macromolecules for closing the gap between the present ineffective treatment and the future effective personalized therapy due to their potential versatile biological properties.

Monogenic diabetes prevalence among Polish children-Summary of 11 years-long nationwide genetic screening program.

BACKGROUND: Estimated monogenic diabetes (MD) prevalence increases as screening programs proceeds. OBJECTIVE: To estimate prevalence of MD among Polish children. SUBJECTS: Patients and their family members suspected of suffering from MD (defined as causative mutation in one of the Maturity Onset Diabetes of the Young or permanent neonatal diabetes mellitus genes) were recruited between January 2005 and December 2015. METHODS: Nationwide prevalence was estimated based on data from 6 administrative provinces (out of 16 in Poland) with high referral rates of patients (>10 per 100 000 children). RESULTS: During the analysis, probands from 322 of 788 screened families tested positive yielding a total of 409 children and 299 family members with MD. An average of 70 probands/year were referred. Screening success rate reached 40% over the study period. We estimated the prevalence of MD in 2015 to 7.52/100 000 children (1 in 13 000). The most frequent MODY in this group was GCK- MODY (6.88/100 000). The prevalence estimates increased nearly 2-fold since our report in 2011 (4.4/100 000). However, the figure reached a plateau because of screening saturation in 2014 what was also proven by lowering of the median age of diagnosis lowered in time (R = -0.73, P = .0172) along with shortening of the delay between clinical and genetic diagnosis (R = -0.65, P = .0417). CONCLUSIONS: The screening for childhood MD in Poland reached a plateau phase after 10 years showing a stable prevalence estimate. The true frequency of MD in the overall population may be higher given later onset of reportedly more frequent types of MD than GCK -MODY.

Craniofacial and cutaneous findings in Noonan, Costello and LEOPARD syndromes.

Noonan, Costello and LEOPARD syndromes belong to a family of cardiofaciocutaneous disorders and share common genetic traits. As they are associated with a germline mutation in genes encoding proteins involved in RAS/MAPK, patients suffering from these syndromes are at a greater risk of cancer and abnormal myelopoiesis in infancy. Patients with cardio faciocutaneous syndromes share some clinically overlapping syndromes, therefore differential diagnosis can be problematic. In this paper we aim at demonstrating distinctive craniofacial and cutaneous manifestations of Noonan, Costello and LEOPARD syndromes which can be useful for clinicians who aim at treatment of children with rare diseases.

Serum Metabolic Fingerprinting Identified Putatively Annotated Sphinganine Isomer as a Biomarker of Wolfram Syndrome.

Wolfram syndrome (WFS) is an example of a rare neurodegenerative disease with coexisting endocrine symptoms including diabetes mellitus as the first clinical symptom. Treatment of WFS is still only symptomatic and associated with poor prognosis. Potential markers of disease progression that could be useful for possible intervention trials are not available. Metabolomics has potential to identify such markers. In the present study, serum fingerprinting by LC-QTOF-MS was performed in patients with WFS (n = 13) and in patients with T1D (n = 27). On the basis of the obtained results, aminoheptadecanediol (17:0 sphinganine isomer) (+50%, p = 0.02), as the most discriminatory metabolite, was selected for validation. The 17:0 sphinganine isomer level was determined using the LC-QQQ method in the samples from WFS patients at two time points and compared with samples obtained from patients with T1D (n = 24) and healthy controls (n = 24). Validation analysis showed higher 17:0 sphinganine isomer level in patients with WFS compared to patients with T1D (p = 0.0097) and control group (p < 0.0001) with progressive reduction of its level after two-year follow-up period. Patients with WFS show a unique serum metabolic fingerprint, differentiating them from patients with T1D. Sphinganine derivate seems to be a marker of the ongoing process of neurodegeneration in WFS patients.

Endothelial integrity may be regulated by a specific antigen via an IgE-mediated mechanism.

BACKGROUND: Human vascular endothelial function and integrity may be regulated by many non-specific factors. However, the potential influence of specific antigens via an IgE-mediated mechanism remains unknown. The aim of the study was to determine the expression of the IgE receptors FcεRI and FcεRII in the human vascular endothelium and to assess their relevance in the IgE-mediated regulation of endothelial integrity. MATERIAL/METHODS: FcεRI and FcεRII expression in human umbilical vein endothelial cells (HUVEC) was genetically assessed by PCR with respective primers and sequencing. HUVEC were cultured with IL-4, and changes in FcεRI and FcεRII mRNA expression were analyzed by real-time PCR. Changes in the integrity of endothelium pre-treated with anti-BSA-DNP IgE following exposure to the specific BSA-DNP antigen was assessed using the Real-time Cell Electric Impedance Sensing system (RTCA-DP). RESULTS: PCR and sequencing revealed the expression of FcεRI and FcεRII receptors in the human vascular endothelium. IL-4 caused respective 2- and 3-fold increases in FcεRI and FcεRII mRNA expression. Exposure of endothelium pre-treated with anti-BSA-DNP IgE to specific BSA-DNP antigen led to a 20% increase of endothelial integrity (p<0.05) after 24 hours, but only in cells pre-incubated with IL-4. CONCLUSIONS: The presence of FcεRI and FcεRII may allow the human vascular endothelium to respond to a specific antigen by increasing its integrity via an IgE-mediated mechanism.

Differential regulation of serum microRNA expression by HNF1ß and HNF1α transcription factors.

AIMS/HYPOTHESIS: We aimed to identify microRNAs (miRNAs) under transcriptional control of the HNF1ß transcription factor, and investigate whether its effect manifests in serum. METHODS: The Polish cohort (N = 60) consisted of 11 patients with HNF1B-MODY, 17 with HNF1A-MODY, 13 with GCK-MODY, an HbA1c-matched type 1 diabetic group (n = 9) and ten healthy controls. Replication was performed in 61 clinically-matched British patients mirroring the groups in the Polish cohort. The Polish cohort underwent miRNA serum level profiling with quantitative real-time PCR (qPCR) arrays to identify differentially expressed miRNAs. Validation was performed using qPCR. To determine whether serum content reflects alterations at a cellular level, we quantified miRNA levels in a human hepatocyte cell line (HepG2) with small interfering RNA knockdowns of HNF1α or HNF1ß. RESULTS: Significant differences (adjusted p < 0.05) were noted for 11 miRNAs. Five of them differed between HNF1A-MODY and HNF1B-MODY, and, amongst those, four (miR-24, miR-27b, miR-223 and miR-199a) showed HNF1B-MODY-specific expression levels in the replication group. In all four cases the miRNA expression level was lower in HNF1B-MODY than in all other tested groups. Areas under the receiver operating characteristic curves ranged from 0.79 to 0.86, with sensitivity and specificity reaching 91.7% (miR-24) and 82.1% (miR-199a), respectively. The cellular expression pattern of miRNA was consistent with serum levels, as all were significantly higher in HNF1α- than in HNF1ß-deficient HepG2 cells. CONCLUSIONS/INTERPRETATION: We have shown that expression of specific miRNAs depends on HNF1ß function. The impact of HNF1ß deficiency was evidenced at serum level, making HNF1ß-dependent miRNAs potentially applicable in the diagnosis of HNF1B-MODY.

Chromosome 18q deletion syndrome with autoimmune diabetes mellitus: putative genomic loci for autoimmunity and immunodeficiency.

A girl with 18q deletion syndrome was diagnosed with autoimmune diabetes mellitus and Hashimoto's thyroiditis at the age of 3 yr. In addition, the girl suffered from recurrent infections due to immunoglobulin A and IgG4 deficiency. She was also found to have CD3+CD4+FoxP3+, CD3+CD4+FoxP3+CD25+, and CD3+CD4+CD25+CD127 regulatory T cells deficiency. The exceptional coincidence of the two autoimmune disorders occurring at an early age, and associated with immune deficiency, implies that genes located on deleted 19.4 Mbp region at 18q21.32-q23 (chr18:58,660,699-78,012,870) might play a role in the pathogenesis of autoimmunity leading to ß cell destruction and diabetes.