Molecular Biomarkers for the Diagnosis, Prognosis, and Pharmacodynamics of Spinal Muscular Atrophy.

Spinal muscular atrophy (SMA) is a progressive degenerative illness that affects 1 in every 6 to 11,000 live births. This autosomal recessive disorder is caused by homozygous deletion or mutation of the SMN1 gene (survival motor neuron). As a backup, the SMN1 gene has the SMN2 gene, which produces only 10% of the functional SMN protein. Nusinersen and risdiplam, the first FDA-approved medications, act as SMN2 pre-mRNA splicing modifiers and enhance the quantity of SMN protein produced by this gene. The emergence of new therapies for SMA has increased the demand for good prognostic and pharmacodynamic (response) biomarkers in SMA. This article discusses current molecular diagnostic, prognostic, and pharmacodynamic biomarkers that could be assessed in SMA patients' body fluids. Although various proteomic, genetic, and epigenetic biomarkers have been explored in SMA patients, more research is needed to uncover new prognostic and pharmacodynamic biomarkers (or a combination of biomarkers).

Genetic and Clinical Characterization of Patients with HNF1B-Related MODY in Croatia.

BACKGROUND: Mutation of the gene encoding Hepatocyte Nuclear transcription Factor-1 Beta (HNF1B) causes a rare monogenetic subtype of Maturity-Onset Diabetes of the Young (MODY). HNF1B-related MODY results in the dysfunction of multiple organ systems. However, genetic analysis enables personalized medicine for patients and families. AIMS: To understand the clinical characteristics and explore the gene mutations in Croatian patients. METHODS: This was a retrospective observational study of individuals (and their relatives) who were, due to the clinical suspicion of MODY, referred to the Department of Laboratory Diagnostics at the University Hospital Centre Zagreb for genetic testing. RESULTS: A total of 118 participants, 56% females, were screened. Seven patients (three females) from five families were identified to have HNF1B-related MODY. The median age at diagnosis was 31 (11-45) years, the median c-peptide was 0.8 (0.55-1.39) nmol/L, the median HbA1c was 9.1 (5.7-18.4)%, and the median BMI was 22.9 kg/m2 (17-24.6). Patients had a variety of clinical manifestations; kidney disease was not as frequent as liver lesions, neuropsychiatric symptoms, hyperlipidemia, hyperuricemia, and hypomagnesemia. We identified two new pathogenic mutations (c.1006C > G protein p.His336Asp on exon 4 and c.1373T > G p protein Val458Gly on exon 7). CONCLUSIONS: In a study involving Croatian patients, new genetic (two previously unknown mutations) and clinical (diverse range of clinical presentations) aspects of HNF1B-related MODY were found.

Total tau in cerebrospinal fluid detects treatment responders among spinal muscular atrophy types 1-3 patients treated with nusinersen.

AIMS: Considering the substantial variability in treatment response across patients with spinal muscular atrophy (SMA), reliable markers for monitoring response to therapy and predicting treatment responders need to be identified. The study aimed to determine if measured concentrations of disease biomarkers (total tau protein, neurofilament light chain, and S100B protein) correlate with the duration of nusinersen treatment and with scores obtained using functional scales for the assessment of motor abilities. METHODS: A total of 30 subjects with SMA treated with nusinersen between 2017 and 2021 at the Department of Pediatrics, University Hospital Centre Zagreb, Croatia, were included in this study. Cerebrospinal fluid (CSF) samples were collected by lumbar puncture prior to intrathecal application of nusinersen. Protein concentrations in CSF samples were determined by enzyme-linked immunosorbent assay in 26 subjects. The motor functions were assessed using functional motor scales. RESULTS: The main finding was significantly decreased total tau correlating with the number of nusinersen doses and motor improvement in the first 18-24 months of treatment (in all SMA patients and SMA type 1 patients). Neurofilament light chain and S100B were not significantly changed after administration of nusinersen. CONCLUSIONS: The measurement of total tau concentration in CSF is a reliable index for monitoring the biomarker and clinical response to nusinersen therapy in patients with SMA.

Is Apolipoprotein E ε2 Associated with Delayed Onset of Non-Lesional Temporal Lobe Epilepsy?

The aim of the study was to evaluate the possible association between Apo E polymorphisms and age at seizure onset in patients with non-lesional temporal lobe epilepsy. Eighty patients with non-lesional temporal lobe epilepsy with or without bilateral tonic-clonic propagation were analyzed. Age at seizure onset was defined as age at the first unequivocal seizure (excluding febrile convulsions). ApoE alleles were determined by a procedure where genome DNA was amplified by chain reaction along with polymerase, using the LightCycler kit (Roche) for ApoE mutations on codons 112 and 158. There was a statistically significant difference between the groups of patients with ApoE ε2/3 and ε3/4 genotypes (p=0.03), but not between patients with ApoE, ε2/3 and ε3/3, and those with ApoE ε3/4 and ε3/3. In conclusion, the results of our study suggested positive association of a specific ApoE genotype and onset of non-lesional temporal lobe epilepsy.

Correction: A European Spectrum of Pharmacogenomic Biomarkers: Implications for Clinical Pharmacogenomics.

[This corrects the article DOI: 10.1371/journal.pone.0162866.].

Is there any association of apolipoprotein E gene polymorphisms with metabolic syndrome in a young population of Croatian origin?

BACKGROUND: Apolipoprotein E has an important role in lipid metabolism and adipocyte activity and apo E gene (APOE) might serve as a potential determinant of metabolic syndrome (MetS). AIM: The aim of the presented study was to investigate the association between APOE polymorphism and MetS in young adult subjects of Croatian origin. METHODS: This study measured biochemical and anthropometric parameters of 149 young (aged 20-33) subjects. The APOE was genotyped by real-time PCR. RESULTS: No APOE genotype significantly increased the risk for development of MetS. Significant association was found between APOE polymorphism and elevated blood pressure (EBP) (p = .019). The carriers of the ɛ4 allele had decreased risk for EBP (OR = 0.28, 95% CI) compared to ɛ3 allele carriers (ɛ3 allele vs others, χ2 = 7.08; p = .005). APOE alleles were significantly associated with the concentration of TC and LDL-C (χ2 = 12.11, p = .002 and χ2 = 15.76, p < .001, respectively). With diet as a modification covariate there was a significant correlation of APOE alleles with the concentrations of adiponectin and leptin (χ2 = 7.076; p = .029 and χ2 = 7.46; p = .024, respectively). CONCLUSION: Although APOE variants were not confirmed as the risk factor for development of MetS, the APOE alleles were associated with some of the metabolic parameters in young Croatian subjects. The relation of APOE alleles with a concentration of adiponectin and leptin depends on the diet intake.

ATP7B Gene Mutations in Croatian Patients with Wilson Disease.

AIMS: Wilson disease (WD) is an autosomal recessive disorder of copper metabolism, characterized by its accumulation in tissues which results in hepatic, neurological, and/or psychiatric symptoms. The aim of this study was to investigate the genetics of WD in Croatian patients. METHODS: Correlation of the clinical presentation subtype and the age at onset of the diagnosis of WD with the ATP7B genotype was investigated in a group of Croatian WD patients. DNA from peripheral blood samples was tested for the p.His1069Gln by direct mutational analysis and other polymorphisms were identified by sequence analysis of coding and flanking intronic regions of ATP7B gene. RESULTS: In the group of 75 WD patients of Croatian origin, 18 different mutations in ATP7B gene were detected, three of which were novel. The p.His1069Gln mutation was most frequent, being detected in 44 Croatian WD patients (58.7%). Most ATP7B mutations (90.4%) were located in exons 5, 8, 13, 14, and 15. CONCLUSIONS: Clinical diagnosis of WD was confirmed in 59 patients by detecting mutations on both ATP7B alleles. The age at onset of WD and the type of WD clinical presentation showed no significant correlation with the ATP7B genotype.

Angiotensin-converting enzyme genotype is not a significant genetic risk factor for idiopathic nephrotic syndrome in Croatian children.

AIM: The association of the angiotensin-converting enzyme (ACE) gene insertion/deletion (I/D) polymorphism with idiopathic nephrotic syndrome (INS) is controversial. Only scarce information on European populations is available. The aim of the study was to investigate the distribution of the ACE gene I/D polymorphism and its impact on INS in children from Croatia. MATERIALS AND METHODS: Ninety-five children with INS were investigated: 30 with minimal change disease (MCD), 35 with mesangial proliferative glomerulonephritis (MesPGN) and 30 with focal segmental glomerulosclerosis (FSGS). The control group consisted of 73 healthy adults. ACE gene was analyzed using the PCR method. The results were correlated with clinical features, renal morphology and response to immunosuppresive therapy. RESULTS: There was no correlation of ACE genotype with gender, age of the disease onset, level of proteinuria, presence of hematuria or hypertension, and GFR at onset of the disease. No statistically significant differences in ACE genotype or allele frequencies between the controls and whole group of patients, MCD group, MesPGN group, FSGS group, steroid sensitive (SS) patients, steroid resistant (SR) patients, as well as each other, were found, although DD genotype tended to be more frequent in FSGS patients, SR patients, and frequent relapsers. Among 11 children treated with cyclophosphamide the D allele was significantly higher among non-responders (p = 0.003). CONCLUSION: DD genotype is not a genetic risk factor for acquiring INS nor significant phenotype modifier regarding to clinical and pathohistological picture and response to steroids in Croatian children. The potential application of ACE genotyping in predicting cyclophosphamide response deserves further investigation.

Estrogen receptor 1 gene (TA)n polymorphism is associated with lone atrial fibrillation in men.

AIM: To determine the association between the number of thymine-adenine (TA)n dinucleotide repeats in the promoter region of the gene coding for the estrogen receptor alpha (ESR1) and the prevalence of lone atrial fibrillation (AF) in men. METHODS: We conducted a case-control study involving 89 men with lone AF and 166 healthy male controls. The ESR1 genotype was established by polymerase chain reaction and capillary electrophoresis. To assess the association of ESR1 genotype with AF, logistic regression models were built with AF as outcome. RESULTS: Men with lone AF had significantly greater number of (TA)n repeats of single alleles than controls (mean ± standard deviation, 19.2 ± 4.2 vs 18 ± 4.3, P = 0.010). After adjustment for other factors, a unit-increase in (TA)n repeat number was associated with a significantly greater likelihood of AF (odds ratio 1.069; 95% confidence interval 1.024-1.116, P=0.002). CONCLUSIONS: Our results indicate that a greater number of (TA)n repeats in the promoter region of ESR1 is associated with a significantly increased likelihood of lone atrial fibrillation in men.

Interaction of genetic risk factors confers increased risk for metabolic syndrome: the role of peroxisome proliferator-activated receptor γ.

AIM: The aim of the study was to estimate the influence of interactions between peroxisome proliferator-activated receptor γ (PPARγ) and target genes lipoprotein lipase (LPL), interleukin 6 (IL6), angiotensin converting enzyme (ACE), and angiotensin II type 1 receptor (AT1R) on metabolic syndrome (MetSy) and its traits. METHODS: The study included 527 participants (263 with MetSy and 264 controls). Genotyping of PPARγ Pro12Ala, LPL PvuII (-/+), IL6 -174G>C, ACE I/D and AT1R 1166A>C was performed using polymerase chain reaction-restriction fragment length polymorphism-based methods. RESULTS: Interaction between PPARγ Pro12Ala and LPL Pvu(-/+) improved prediction of MetSy over and above prediction based on a model containing no interactions (χ(2)=7.22; df=1; p=0.007). In the group of participants with PPARγ Pro12Ala or Ala12Ala genotypes, those with the LPL Pvu (-/+) or (+/+) genotype had greater odds for MetSy (odds ratio OR=5.98; 95% confidence interval CI: 1.46-24.47, p=0.013). Interaction between PPARγ Pro12Ala and IL6 -174G>C improved prediction of high fasting blood glucose (χ(2)=13.99; df=1; p<0.001). PPARγ Ala12 variant was found protective in patients with IL6 -174GG genotype (OR=0.10; 95% CI: 0.02-0.57, p=0.01), while in the case of IL6 -174C allele carriers, for PPARγ Ala12 carriers, larger odds for high glucose levels compared with Pro12 variant were observed (OR=2.39; 95% CI: 1.11-5.17, p=0.026). Interactions of PPARγ and ACE were significant for BMI. In the group with ACE DD genotype, those with PPARγ Pro12Ala or Ala12Ala genotype have greater odds for obesity (OR=9.98; 95% CI: 1.18-84.14, p=0.034). CONCLUSIONS: PPARγ gene variants can, in interaction with some of its target genes, modulate physiological processes leading to the development of MetSy.

Effects of lipoprotein lipase and peroxisome proliferator-activated receptor-gamma gene variants on metabolic syndrome traits.

Peroxisome proliferator activated receptor-gamma (PPARG) and lipoprotein lipase (LPL) play important role in lipid homeostasis, insulin resistance and adipogenesis, and their gene variability could be considered as predictive genetic markers for metabolic syndrome (MetSy). The aim of the study was to estimate possible associations of PPARG (Pro12Ala) and LPL PvuII (+/-) polymorphisms with MetSy and its traits. Study included 527 subjects. According to the modified National Cholesterol Education Program Adult Treatment Panel III definitions, subjects were classified into the metabolic syndrome group and control group. Genotyping was performed using polymerase chain reaction-restriction fragment length polymorphism methods. In the total sample, LPL variants were associated with waist circumference (chi2 = 7.263, d.f = 2, p = 0.026) and with BMI (chi2 = 6.549, d.f = 2, p = 0.038), where PvuII (+/+) genotype carriers had the highest risk for increased waist circumference (specific PvuII (+/+) vs. others analysis chi2 = 7.033, p = 0.008) and increased BMI (specific PvuII( +/+) vs. others analysis chi2 = 5.154, p = 0.023). LPL gene variants were also associated with HDL-C levels (chi2 = 6.901, d.f = 2, p = 0.032), where PvuII (-/-) genotype carriers had higher HDL-C values in comparison to others (specific Pvu (+/+) vs. others analysis chi2 = 6.504, p = 0.011). Furthermore, PvuII (-) allele carriers had significantly lower glucose (allele based analysis Add Value = -0.0878, chi2 = 5.878, d.f. = 1, p = 0.015). Significant interaction was detected between PPARG and LPL that affected HDL-C levels in male population (chi2 = 11.790, d.f = 1, p = 0.0006) in the manner that Ala/PvuII(+) contributed to the lowest HDL-C values (Specific Ala/ Pvu(+) vs. others analysis was chi2 = 11.750, p = 0.0006). According to obtained results LPL and PPARG gene variants could be susceptibility factors of obesity and lipid status, contributing to development of MetSy, particularly in males. Because of antiatherogenic function of HDL-C, the identification of genetic variants associated with HDL-C can provide useful information related to genotype-phenotype relationships. Since the interplay between PPARG and LPL gene and gender seems to be significant it could point to the personalized behavioural recommendations for prevention of metabolic and cardiovascular diseases.

ZMYND10 is mutated in primary ciliary dyskinesia and interacts with LRRC6.

Defects of motile cilia cause primary ciliary dyskinesia (PCD), characterized by recurrent respiratory infections and male infertility. Using whole-exome resequencing and high-throughput mutation analysis, we identified recessive biallelic mutations in ZMYND10 in 14 families and mutations in the recently identified LRRC6 in 13 families. We show that ZMYND10 and LRRC6 interact and that certain ZMYND10 and LRRC6 mutations abrogate the interaction between the LRRC6 CS domain and the ZMYND10 C-terminal domain. Additionally, ZMYND10 and LRRC6 colocalize with the centriole markers SAS6 and PCM1. Mutations in ZMYND10 result in the absence of the axonemal protein components DNAH5 and DNALI1 from respiratory cilia. Animal models support the association between ZMYND10 and human PCD, given that zmynd10 knockdown in zebrafish caused ciliary paralysis leading to cystic kidneys and otolith defects and that knockdown in Xenopus interfered with ciliogenesis. Our findings suggest that a cytoplasmic protein complex containing ZMYND10 and LRRC6 is necessary for motile ciliary function.

Signal hyperintensities on brain magnetic resonance imaging in patients with primary Sjögren syndrome and frequent episodic tension-type headache: relation to platelet serotonin level and disease activity.

OBJECTIVE: To examine differences in number and size of signal hyperintensities (SH) on magnetic resonance imaging (MRI) between patients with primary Sjögren syndrome (pSS) and controls who all had frequent episodic tension-type headache (FETH), and to investigate their relation to platelet serotonin level (PSL), patient age, disease duration, and activity. METHODS: SH in 22 pSS patients with FETH were compared to 20 aged-matched controls with FETH, using the modified semiquantitative rating scale. Spectrofluorimetry was used for determination of PSL, and the European League Against Rheumatism SS Disease Activity Index (ESSDAI) for disease activity assessment. RESULTS: Statistically significant differences in the total number of SH were noted infratentorially (p = 0.025) and in the basal ganglia for lesions of diameter > 5 mm (p = 0.048). Significant correlations were found between disease duration and number of overall lesions > 5 mm (p = 0.04) and subcortical lesions of diameter 2-5 mm (p = 0.035). Number of periventricular SH inversely correlated to PSL (p = 0.019) and to patient age (p = 0.004), without association with markers of immunoinflammation and ESSDAI. CONCLUSION: Our study showed that SH on brain MRI are more common in specific regions of the brain in pSS patients with FETH than in controls with FETH, signifying a more widespread cerebral vasculopathy in SS patients with FETH. Periventricular SH seem to be associated to increased platelet serotonin release in pSS patients with FETH and correlated with disease duration, without correlation to the actual ESSDAI and markers of immunoinflammation, and might be linked with chronic immunoinflammation of low-grade intensity and vasculitis in pSS.

Mutation detection in Croatian patients with familial hypercholesterolemia.

Familial hypercholesterolemia (FH) is caused by mutations in the genes for LDLR, APOB or PCSK9, and identification of the causative mutation provides definitive diagnosis so that the patient can be treated, their relatives tested and, therefore, premature heart disease prevented. DNA of eight unrelated individuals with clinically diagnosed FH were analyzed using a High-Resolution Melting method (HRM) for the LDLR gene (coding region, promoter and intron/exon boundaries), the APOB gene (part exon 26) and the PCSK9 gene (exon7). Variations found were sequenced and the effect on function of confirmed variants examined using predictive algorithms. Gross deletions and insertions were analysed using MLPA. Three novel LDLR variants were found, p.(S470C), p.(C698R) and c.2312-2A>C. All were predicted to be pathogenic using predictive algorithms. Three previously reported disease-causing mutations were identified (p.(G20R), p.(N272T) and p.(S286R); the latter was also carried by a hypercholesterolaemic relative. One patient carried the pathogenic APOB variant p.(R3527Q). No large LDLR deletions nor insertions were found, neither were any PCSK9 variants identified. HRM is a sensitive method for screening for mutations. While the causative mutation has been identified in 88% of these clinically defined FH patients, there appears to be a high degree of allelic heterogeneity in Croatian patients.

Reconstructing the population history of European Romani from genome-wide data.

The Romani, the largest European minority group with approximately 11 million people, constitute a mosaic of languages, religions, and lifestyles while sharing a distinct social heritage. Linguistic and genetic studies have located the Romani origins in the Indian subcontinent. However, a genome-wide perspective on Romani origins and population substructure, as well as a detailed reconstruction of their demographic history, has yet to be provided. Our analyses based on genome-wide data from 13 Romani groups collected across Europe suggest that the Romani diaspora constitutes a single initial founder population that originated in north/northwestern India ~1.5 thousand years ago (kya). Our results further indicate that after a rapid migration with moderate gene flow from the Near or Middle East, the European spread of the Romani people was via the Balkans starting ~0.9 kya. The strong population substructure and high levels of homozygosity we found in the European Romani are in line with genetic isolation as well as differential gene flow in time and space with non-Romani Europeans. Overall, our genome-wide study sheds new light on the origins and demographic history of European Romani.

Adiponectin level and gene variability are obesity and metabolic syndrome markers in a young population.

BACKGROUND AND AIMS: Human obesity is accepted as an important risk factor for development of MetS. Adiponectin is linked to central obesity and ADIPOQ variants are promising markers for understanding the genetic base of obesity-related disorders. We performed analyses of adiponectin concentrations and ADIPOQ variants and tested their associations with obesity and MetS in young subjects of Croatian origin. METHODS: Biochemical and anthropometric parameters of MetS were obtained for 149 unrelated subjects. Adiponectin levels were measured by ELISA assay. ADIPOQ -11391G>A and -11377C>G were genotyped by real-time PCR. RESULTS: BMI and WC, TG and GLUC showed inverse correlation, whereas HDL-C showed a positive correlation with adiponectin concentrations. For central obesity, we found association with -11377C>G and with -11391G>A polymorphisms. ADIPOQ -11377GG and -11391GA significantly increased the risk for the development of central obesity (OR 5.57 and OR 3.37, respectively). Significant association was found between -11391A, -11377G allele and haplotype and increased TG. -11377C>G and -11391G>A variant were significantly associated with the incidence of MetS. C>G mutation at position -11377 significantly increased the risk of MetS development (OR = 2.93). Compared with the -11391G homozygotes, carriers of the A allele had a significantly increased risk for the development of MetS (OR = 3.15). The test of overall association showed a statistically significant correlation of MetS with -11377C>G and -11391G>A haplotypes (p = 0.008). CONCLUSIONS: Analysis of adiponectin concentration and ADIPOQ -11391G>A and -11377C>G gene variants may be clinically meaningful for estimation of MetS risk in a young population.