MiRNA Signatures Related to Invasiveness and Recurrence in Patients With Non-Functioning Pituitary Neuroendocrine Tumors.

PURPOSE: This preliminary study aimed to analyze and identify differentially expressed miRNAs in Bulgarian patients with non-functioning pituitary neuroendocrine tumors (NFPitNET). The relationship between deregulated miRNAs and tumor invasiveness, recurrence, and size was determined. METHODS: Twenty patients with NFPitNET were selected and fresh pituitary tumor tissues were collected. RNA containing miRNAs were isolated using miRNAeasy mini kit and analyzed by quantitative real-time polymerase chain reaction (PCR) using LNA miRNA Cancer-Focus PCR Panel (Qiagen). RESULTS: Three miRNAs (miR-210-3p, miR-149-3p, and miR-29b-3p) were deregulated in invasive compared to non-invasive NFPitNETs. Differential expression of four-miRNA signatures - miRNA-17, miR-19, miR-106a, and miR-20, correlated with patient recurrence. CONCLUSION: This prospective pilot study selected a unique miRNA expression profile, that correlates with invasiveness and recurrence in non-functioning pituitary neuroendocrine tumors. Moreover, some of the selected miRNAs are reported for the first time in patients with this disease, shedding light on the molecular mechanisms involved in pituitary pathogenesis. The identified miRNAs demonstrate potential as biomarkers, deserving further investigation in a larger cohort to validate their clinical applicability.

Melatonin Receptor 1B and Corticosteroid Receptor Polymorphisms in Infertile Women with Implantation Failure and Miscarriages.

BACKGROUND: The development of assisted reproductive techniques has significantly improved fertility chances in many women, but recurrent implantation failure (RIF) and miscarriages (RM) might preclude successful pregnancy. Alterations in the intrinsic secretory patterns of melatonin and cortisol influence reproduction in humans, and imperfection of receptor - dependent signaling may additionally compromise the hormonal effects. Therefore, the present study aims to investigate the influence of certain melatonin and cortisol receptor polymorphisms in infertile women. METHODS: A total of 111 female infertile patients suffering from implantation failure and/or miscarriages were genotyped for MTNR1B rs1562444, MTNR1Brs10830962, GCCR rs41423247, and GCCR ER22/23EK variants. Additionally, 106 female volunteers were genotyped for the same polymorphisms. RESULTS: The allele and genotype distribution of the investigated polymorphisms did not differ between infertile women and the control group. Significantly more women with history of RIF have MTNR1B rs1562444 G-allele-containing genotypes in comparison to AA carriers (19.3% vs. 3.6%, p = 0.004). The minor allele of the ER22/23EK variant was more frequent in infertile patients with three or more unsuccessful implantation attempts than in other women (12.5% vs. 2.4%, p = 0.025). CONCLUSIONS: Melatonin receptor 1B polymorphisms might affect embryo implantation and early pregnancy loss, while their influence on late pregnancy complications needs further evaluation. The possible association between the cortisol receptor ER22/23EK variant and recurrent implantation failure might help to differentiate women who could benefit from corticosteroid treatment.

Blood Plasma Calorimetric Profiles of Women with Preeclampsia: Effect of Oxidative Stress.

Preeclampsia is a pregnancy-related disease with poor placentation and presents itself through hypertension and proteinuria. The disease is also associated with the oxidative modification of proteins in maternal blood plasma. In this work, we combine differential scanning calorimetry (DSC), capillary electrophoresis, and atomic force microscopy (AFM) to evaluate the changes in the plasma denaturation profiles of patients with preeclampsia (PE) as compared with those of pregnant controls. Our results demonstrate that the last trimester of pregnancy substantially affects the main calorimetric characteristics of blood plasma from pregnant controls relative to nonpregnant women. These variations correlate well with the changes in protein levels determined by electrophoresis. DSC analysis revealed significant deviations in the plasma heat capacity profiles of preeclamptic patients from those of pregnant controls. These alterations are expressed mainly in a substantial reduction in albumin-assigned transitions and an upward shift in its denaturation temperature, lower calorimetric enthalpy changes, and a reduced ratio of heat capacity in the albumin/globulin-assigned thermal transitions, which are more pronounced in severe PE cases. The in vitro oxidation model shows that the alteration of PE thermograms is partly related to protein oxidation. AFM data detected numerous aggregate formations in the plasma of PE samples and fewer small ones in the pregnant controls, which are not found in healthy nonpregnant samples. These findings could serve as a basis for further investigations to reveal the possible relationship between albumin thermal stabilization, the increased inflammatory state and oxidative stress, and protein misfolding in preeclampsia.

Hypomyelination with Atrophy of Basal Ganglia and Cerebellum (HABC) Due to UFM1 Mutation in Roma Patients - Severe Early Encephalopathy with Stridor and Severe Hearing and Visual Impairment. A Single Center Experience.

BACKGROUND: Hypomyelination with atrophy of the basal ganglia and cerebellum (H-ABC) is a neurodegenerative disease with neurodevelopmental delay, motor, and speech regression, pronounced extrapyramidal syndrome, and sensory deficits due to TUBB4A mutation. In 2017, a severe variant was described in 16 Roma infants due to mutation in UFM1. OBJECTIVE: The objective of this study is to expand the clinical manifestations of H-ABC due to UFM1 mutation and suggest clues for clinical diagnosis. METHODOLOGY: Retrospective analysis of all 9 cases with H-ABC due to c.-273_-271delTCA mutation in UFM1 treated during 2013-2020 in a Neuropediatric Ward in Plovdiv, Bulgaria. RESULTS: Presentation is no later than 2 months with inspiratory stridor, impaired sucking, swallowing, vision and hearing, and reduced active movements. By the age of 10 months, a monomorphic disease was observed: microcephaly (6/9), malnutrition (5/9), muscle hypertonia (9/9) and axial hypotonia (4/9), progressing to opisthotonus (6/9), dystonic posturing (5/9), nystagmoid ocular movements (6/9), epileptic seizures (4/9), non-epileptic spells (3/9). Dysphagia (7/9), inspiratory stridor (9/9), dyspnea (5/9), bradypnea (5/9), apnea (2/9) were major signs. Vision and hearing were never achieved or lost by 4-8 mo. Neurodevelopment was absent or minimal with subsequent regression after 2-5 mo. Brain imaging revealed cortical atrophy (7/9), atrophic ventricular dilatation (4/9), macrocisterna magna (5/9), reduced myelination (6/6), corpus callosum atrophy (3/6) and abnormal putamen and caput nuclei caudati. The age at death was between 8 and 18 mo. CONCLUSION: Roma patients with severe encephalopathy in early infancy with stridor, opisthotonus, bradypnea, severe hearing and visual impairment should be tested for the Roma founder mutation of H-ABC in UFM1.

Altered Thermal Behavior of Blood Plasma Proteome Related to Inflammatory Cytokines in Early Pregnancy Loss.

Early pregnancy loss (EPL) is a relatively common pathology of which almost 50% of cases remain idiopathic. In the search for novel biomarkers, differential scanning calorimetry (DSC) is intensively used to characterize the thermodynamic behavior of blood plasma/serum proteome in health and disease. Herein, for the first time, we investigate the DSC denaturation profiles of blood plasma derived from patients suffering EPL compared to healthy pregnant and non-pregnant women. Data analysis reveals that 58% of the EPL thermograms differ significantly from those of healthy pregnant women. Thermal stabilization of a fraction of albumin-assigned transition with concomitant suppression of the major and enhancement of the globulin-assigned transition are characteristic features of EPL calorimetric profiles that could be used as a new indicator of a risk pregnancy. The presented results suggest an altered composition or intermolecular interactions of the plasma proteome of women with EPL. In addition, the alterations of the EPL thermograms correlate with the increased blood levels of tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6) and a higher prevalence of the polymorphism in the plasminogen activator inhibitor type-1 (PAI-1) gene, suggesting an expression of an overall enhanced immune response. The concomitant changes in plasma thermograms confirm the potential of the DSC approach for distinguishing changes in the pathological state of the blood plasma proteome.

Retrospective Diagnosis of Pontocerebellar Hypoplasia Type 1B in a Family with Two Deceased Newborn Children.

Pontocerebellar hypoplasia type 1B is a severe autosomal recessive neurologic disorder characterized by a combination of cerebellar and spinal motor neuron degeneration beginning at birth. Pontocerebellar hypoplasia type 1B is caused by mutations in EXOSC3 gene. High prevalence of the p.Gly31Ala mutation was found recently, especially in the Roma ethnic minority. We present a young Bulgarian Roma family with two deceased newborn children manifesting severe neuromuscular disorder including severe muscle weakness, respiratory distress, and multiple joint contractures. Based on the clinical signs and family's population characteristics, DNA testing for the previously described EXOSC3 in Bulgarian Roma mutation c.92G > C; p.Gly31Ala was performed on blood samples of both parents and they were found to be heterozygous carriers. This finding indirectly confirmed the diagnosis of pontocerebellar hypoplasia type B in the deceased offspring. Knowledge of population-specific molecular bases of genetic conditions was the key to final diagnosis in the presented family. Designing of population-based clinical-genetic panels may be a powerful diagnostic tool for patients with such origin. Preconception carrier screening in high-risk population groups is a feasible option to discuss.

Restriction of the Global IgM Repertoire in Antiphospholipid Syndrome.

The typical anti-phospholipid antibodies (APLA) in the anti-phospholipid syndrome (APS) are reactive with the phospholipid-binding protein ß2GPI as well as a growing list of other protein targets. The relation of APLA to natural antibodies and the fuzzy set of autoantigens involved provoked us to study the changes in the IgM repertoire in APS. To this end, peptides selected by serum IgM from a 7-residue linear peptide phage display library (PDL) were deep sequenced. The analysis was aided by a novel formal representation of the Igome (the mimotope set reflecting the IgM specificities) in the form of a sequence graph. The study involved women with APLA and habitual abortions (n=24) compared to age-matched clinically healthy pregnant women (n=20). Their pooled Igomes (297 028 mimotope sequences) were compared also to the global public repertoire Igome of pooled donor plasma IgM (n=2 796 484) and a set of 7-mer sequences found in the J regions of human immunoglobulins (n=4 433 252). The pooled Igome was represented as a graph connecting the sequences as similar as the mimotopes of the same monoclonal antibody. The criterion was based on previously published data. In the resulting graph, identifiable clusters of vertices were considered related to the footprints of overlapping antibody cross-reactivities. A subgraph based on the clusters with a significant differential expression of APS patients' mimotopes contained predominantly specificities underrepresented in APS. The differentially expressed IgM footprints showed also an increased cross-reactivity with immunoglobulin J regions. The specificities underexpressed in APS had a higher correlation with public specificities than those overexpressed. The APS associated specificities were strongly related also to the human peptidome with 1 072 mimotope sequences found in 7 519 human proteins. These regions were characterized by low complexity. Thus, the IgM repertoire of the APS patients was found to be characterized by a significant reduction of certain public specificities found in the healthy controls with targets representing low complexity linear self-epitopes homologous to human antibody J regions.

Morphometric and Nanomechanical Features of Erythrocytes Characteristic of Early Pregnancy Loss.

Early pregnancy loss (EPL) is estimated to be between 15 and 20% of all adverse pregnancies. Approximately, half of EPL cases have no identifiable cause. Herein, we apply atomic force microscopy to evaluate the alteration of morphology and nanomechanics of erythrocytes from women with EPL with unknown etiology, as compared to healthy pregnant (PC) and nonpregnant women (NPC). Freshly isolated erythrocytes from women with EPL differ in both the roughness value (4.6 ± 0.3 nm, p < 0.05), and Young's modulus (2.54 ± 0.6 MPa, p < 0.01) compared to the values for NPC (3.8 ± 0.4 nm and 0.94 ± 0.2 MPa, respectively) and PC (3.3 ± 0.2 nm and 1.12 ± 0.3 MPa, respectively). Moreover, we find a time-dependent trend for the reduction of the cells' morphometric parameters (cells size and surface roughness) and the membrane elasticity­much faster for EPL than for the two control groups. The accelerated aging of EPL erythrocytes is expressed in faster morphological shape transformation and earlier occurrence of spiculated and spherical-shaped cells, reduced membrane roughness and elasticity with aging evolution. Oxidative stress in vitro contributed to the morphological cells' changes observed for EPL senescent erythrocytes. The ultrastructural characteristics of cells derived from women with miscarriages show potential as a supplementary mark for a pathological state.

The role of IL-17 rs2275913, IL-17RC rs708567 and TGFB1 rs1800469 SNPs and IL-17A serum levels in patients with lupus nephritis.

Systemic lupus erythematosus (SLE) is a multifactorial autoimmune disease and polymorphisms in the cytokine genes and their receptors are thought to influence its development. The aim of this case-control study was to investigate the association of the IL-17A rs2275913, IL-17RC rs708567 and TGFB1 rs1800469 polymorphisms with SLE, its clinical manifestations and the polymorphisms influence on the IL-17A serum levels. Altogether 59 SLE patients with lupus nephritis and 95 healthy controls were genotyped by TaqMan assay. Serum levels were determined by Human IL-17A Platinum ELISA kit. From the studied polymorphisms, only TGFB1 T allele was found to be associated with SLE. Within the patient group, IL-17A GG genotype and TGFB1 -509T allele showed an association with the neurological disease and IL-17RC CC genotype appeared to be associated with lupus arthritis. The IL17A serum levels in the SLE and control groups (7.24 pg/ml and 5.76 pg/ml, respectively) did not show any statistical difference. A weak correlation between IL17A levels and SLEDAI-2K was observed. Our results indicate that IL-17A rs2275913, IL-17RCrs708567 and TGFB1 rs1800469 polymorphisms might play a role in the susceptibility and the clinical manifestations of SLE and IL-17A serum levels should be monitored in the course of the disease. The identification of subsets of SLE with an IL-17-driven disease could improve the therapeutic approach leading to more precise personalized treatment.

Current Status of Newborn Screening in Southeastern Europe.

Significant part of Southeastern Europe (with a population of 76 million) has newborn screening (NBS) programs non-harmonized with developed European countries. Initial survey was conducted in 2013/2014 among 11 countries from the region (Albania, Bulgaria, Bosnia and Herzegovina (BIH), Croatia, Kosovo, Macedonia, Moldova, Montenegro, Romania, Serbia, and Slovenia) to assess the main characteristics of their NBS programs and their future plans. Their cumulative population at that time was ~52,5 million. At that time, none of the countries had an expanded NBS program, while phenylketonuria screening was not introduced in four and congenital hypothyroidism in three of 11 countries. We repeated the survey in 2020 inviting the same 11 countries, adding Cyprus, Greece, Hungary, and Malta (due to their geographical position in the wider region). The aims were to assess the current state, to evaluate the change in the period, and to identify the main obstacles impacting the implementation of expanded NBS and/or reaching a wider population. Responses were collected from 12 countries (BIH-Federation of BIH, BIH-Republic of Srpska, Bulgaria, Croatia, Greece, Hungary, Kosovo, North Macedonia, Malta, Montenegro, Romania, Serbia, Slovenia) with a population of 68.5 million. The results of the survey showed that the regional situation regarding NBS only modestly improved in this period. All of the surveyed countries except Kosovo screened for at least congenital hypothyroidism, while phenylketonuria was not screened in four of 12 countries. Croatia and Slovenia implemented an expanded NBS program using tandem mass spectrometry from the time of last survey. In conclusion, the current status of NBS programs in Southeastern Europe is very variable and is still underdeveloped (or even non-existent) in some of the countries. We suggest establishing an international task-force to assist with implementation and harmonization of basic NBS services where needed.

PNPLA3 I148M Polymorphism in Patients with Nonalcoholic Fatty Liver Disease, Obesity and Prediabetes.

BACKGROUND AND AIMS: Nonalcoholic fatty liver disease (NAFLD) is closely associated with obesity and insulin resistance, and therefore predisposes to type 2 diabetes and cardiovascular diseases. Lipid deposition in the liver seems to be critical in the pathogenesis of NAFLD. A common genetic variant, the patatin-like phospholipase domain-containing protein 3 (PNPLA3) has been associated with NAFLD. The aim of the present study was to evaluate the association between PNPLA3, key gene of lipid metabolism and the metabolic traits in obesity NAFLD patients with and without prediabetes. METHODS: A total of 208 obese NAFLD patients without (n=125) and with prediabetes (n=83) were included. The genotyping of PNPLA3 I148M variant (rs738409) was performed by restriction analysis. RESULTS: Regarding rs738409 (I148M) polymorphism, CG genotype was positively correlated with prediabetes, insulin resistance, dyslipidemia and metabolic syndrome compared to the wild CC genotype. The carriers of the PNPLA3 I148M variant have 9.6-fold higher risk of glucose disturbances compared to wild genotype (OR 9.649, 95%CI 2.100-44.328, р=0.004). The carriers of the PNPLA3 I148M variant also have a 3 times higher risk for the presence of metabolic syndrome (OR 2.939, 95% CI: 1.590-5.434, p=0.001) and a 2.1-fold higher risk for the presence of insulin resistance (OR 2.127, 95% CI: 1.078-4.194, p=0.029). CONCLUSIONS: PNPLA3 I148M is associated with increased risk of prediabetes, metabolic syndrome and insulin resistance in obese patients with NAFLD.

Identification of 99% of CFTR gene mutations in Bulgarian-, Bulgarian Turk-, and Roma cystic fibrosis patients.

BACKGROUND: The spectrum and frequencies of CFTR mutations causing Cystic fibrosis (CF) varies among different populations in Europe, and beyond. METHODS: We identified 98.9% of all CFTR mutations in a representative cohort of 140 CF patients comprising 107 Bulgarian- (BG), 17 BG Turk-, and 16 BG Roma cases. The compiled clinical and genotype dataset includes 110 previously analyzed patients with 30 cases currently analyzed for rare CFTR variants by massively parallel sequencing of the entire CFTR coding region and adjacent introns combined with the analysis of intra-CFTR rearrangements. RESULTS: Altogether 53 different mutations, of which 15 newly identified in the BG CF population, were observed. Comparison of clinical and laboratory data between individual BG ethnic groups proved that BG Roma have a more severe nutritional status and are younger than other CF patients, as well as that the spectrum mutations differs between them. CONCLUSION: This collaborative study improves genetic counselling in BG, facilitates introduction of multitier CF neonatal screening and fosters public health measures for improvement of care in the Roma CF population.

Long-term follow-up of a female patient with non-classical 11ß-hydroxylase deficiency and two novel mutations in CYP11B1.

11ß-Hydroxylase deficiency is the second most common enzyme disorder after 21-hydroxylase deficiency causing congenital adrenal hyperplasia (CAH11ß). In females, the clinical phenotype of CAH11ß classic forms is associated with ambiguous genitalia, virilization and hypertension, while most common complaints in milder non-classic forms include hirsutism, acne, menstrual disturbances, and infertility. Herein, we present clinical and genetic characteristics of an adult woman with 11ß-hydroxylase deficiency, hypertension and infertility; she has been followed up from her first pregnancy to her early menopause. Genetic analyses of the patient revealed a compound-heterozygosity due to two variants in the CYP11B1 gene p.Val316Met and p.Asp480ThrfsTer2. Both mutations have not been previously reported as pathogenic in the literature. Emerging questions concerning the clinical management, fertility potential, mineral corticoid abnormalities and perimenopausal transition in patients with non-classic CAH11ß have also been briefly discussed. The presented case of an adult woman with CAH11ß shows that the proper diagnosis and close monitoring of patients with different CAH forms might ensure good therapy adherence and successful fertility.

Positive association between a polymorphic locus near the LBX1 gene and predisposition of idiopathic scoliosis in Southeastern European population.

Idiopathic scoliosis (IS) is a common medical condition in children, characterized by three-dimensional spinal curve and strong evidence of genetic predisposition. The purpose of the present case-control study is to examine the association between the polymorphic variant rs11190870 (T/C), near the LBX1 gene, and IS predisposition in distinct subgroups based on age at onset, family history and gender. A total of 127 IS patients and 254 unrelated controls of Southeastern European descent were recruited. The genotyping was carried out by TaqMan real-time amplification technology. The results were analyzed by the Pearson's Chi-squared Test and the Fisher's Exact Test with a value of p less than 0.05 as statistically significant. The T allele and homozygous TT genotype were associated with a greater incidence of IS. Our results suggest that there is a genetic association with IS in adolescents, familial and non-familial cases, and in females. Larger case-control studies are necessary to examine the genetic factors of IS/AIS etiology in infants, juveniles and males. In conclusion, the molecular genetic identification of diagnostic and prognostic molecular markers would make an early treatment including minimally invasive procedures possible.

Genetically Determined TNF-α Secretion Influences the Development of Dermatomyositis and Systemic Lupus Erythematosus.

BACKGROUND: Abnormal secretion of TNF-α is known to play a role in the pathogenesis of dermatomyositis and systemic lupus erythematosus. MATERIALS AND METHODS: In the present study we have analyzed the concentrations of TNF-α in the sera of 30 patients with systemic lupus erythematosus (SLE), 28 with dermatomyositis (DM) and 30 healthy controls by standard ELISA tests. RESULTS: We have found that -308A allele increases TNF-α secretion, while -1031C and -863A alleles decrease it. The -857C/T and 489G/A polymorphisms appeared in strong linkage disequilibrium (D'=0.93) but they did not seem to affect TNF-α secretion. CONCLUSION: TNF-α polymorphisms play a significant role in its secretion and influence the development of DM and SLE.

Positive Association between TGFB1 Gene and Susceptibility to Idiopathic Scoliosis in Bulgarian Population.

Idiopathic scoliosis (IS) is a common medical condition beginning in childhood and characterized by strong evidence for a genetic susceptibility to three-dimensional spinal deformity. The primary goal of the current case-control study is to examine the association between the TGFB1 (-509C/T) functional polymorphic variant and genetic predisposition to IS in the Bulgarian population and the genotype-phenotype correlations in distinct case-control subgroups based on age at onset, family history, and gender. A total of 127 patients with primary scoliosis and 254 gender-matched control subjects were recruited. The mean Cobb angle was 53.8 ± 21.2°. Genotyping of cases and controls was performed using the TaqMan real-time amplification technique. The results were processed statistically using Pearson's Chi-squared test and Fisher's exact test with a value of p less than 0.05 as statistically significant. The polymorphic T allele and TT genotype were associated with a greater incidence of IS and can be considered as predisposing factors with a moderate effect on deformity development. The current results suggested that there was a genetic predisposition in early and late onset IS and familial, sporadic, and female cases. Nevertheless, replication studies are needed to reveal the relationship between the TGFB1 locus and certain subtypes of IS in different populations.

New Territory for an Old Disease: 5-Alpha-Reductase Type 2 Deficiency in Bulgaria.

Disorders/differences of sexual development (DSD) are a group of conditions, some of which can be clinically indistinguishable mainly due to their phenotypic variability. Defining the molecular basis of their wide spectrum is still in progress. The diagnosis of 5-alpha-reductase type 2 (5α-reductase-2) deficiency is difficult especially in newborns and pre-pubertal individuals, and as a result its frequency might be underestimated. In the present study, we describe the clinical characteristics and molecular defects in 3 nonrelated 5α-reductase-2 deficiency patients of Bulgarian descent. Sequencing analysis revealed the mutations p.Y188CfsX9 and p.G196S, and MLPA analysis showed a deletion of exon 1 in the SRD5A2 gene. The observed genetic substitutions were not detected in 76 additionally screened unrelated controls, but a heterozygous healthy carrier of the p.R171S mutation was found. This is the first study on the molecular basis of 5α-reductase-2 deficiency in Bulgaria. It suggests that the carrier frequency of mutations in the SRD5A2 gene might be noteworthy worldwide. There is no correlation between cultural aspects, location, and/or population size and the number of different mutations in SRD5A2 detected, and more efforts should be made to determine the prevalence of this condition in different geographic areas. Our study supports the importance of genetic testing in 46,XY DSD patients, especially in countries or regions where 5α-reductase-2 deficiency has not been reported so far.

Intragenic Deletions in ATP7B as an Unusual Molecular Genetics Mechanism of Wilson's Disease Pathogenesis.

Wilson's disease (WD) is an autosomal recessive disorder caused by mutations in the ATP7B resulting in copper overload in the liver and brain. Direct sequencing is routinely used to confirm WD diagnosis; however, partial and whole gene deletions in the heterozygous state cannot be detected by exon amplification since the normal allele will mask its presence. The aim of the present work was to search for unusual mutational events in the unexplained WD cases and to provide insight into the mechanisms. Out of 1420 clinically and biochemically confirmed WD samples received between 2000 and 2014 for routine mutation analysis, we were unable to detect mutant alleles in 142 samples, after extensive sequencing analysis. We used selective amplification and MLPA to identify the partial gene deletions and identified three different partial gene deletions in seven different families. All three deletions were fully characterized at the DNA sequence level. We report the first hemizygous case with WD due to intragenic deletion in the ATP7B (c.3134_3556+689del). This novel deletion resulted from an excision event mediated by consensus sequences in an AluSq2 repeat element and could be traced to micro homologous end joining (MMEJ). Finally, we determined the prevalence of the three deletions in DNA samples from a multinational group of WD patients. Our results emphasize the need for searching mutant alleles beyond routine methods and highlight that large ATP7B deletions are rare, but account for a detectable proportion in some WD patients. Screening for gene aberrations will further improve mutation detection in patients with unidentified ATP7B mutations presenting with clinical manifestations of WD.