Clinical Presentation of Left Ventricular Noncompaction Cardiomyopathy and Bradycardia in Three Families Carrying HCN4 Pathogenic Variants.

BACKGROUND: Left ventricular noncompaction (LVNC) is a genetically and phenotypically heterogeneous cardiomyopathy in which myocardium consists of two, distinct compacted and noncompacted layers, and prominent ventricular trabeculations and deep intertrabecular recesses are present. LVNC is associated with an increased risk of heart failure, atrial and ventricular arrhythmias and thromboembolic events. Familial forms of primary sinus bradycardia have been attributed to alterations in HCN4. There are very few reports about the association between HCN4 and LVNC. The aim of our study was to characterize the clinical phenotype of families with LVNC and sinus bradycardia caused by pathogenic variants of the HCN4 gene. METHODS: From March 2008 to July 2021, we enrolled six patients from four families with diagnosed isolated LVNC based on the clinical presentation, family history and echocardiographic and cardiovascular magnetic resonance (CMR) evidence of LVNC. Next generation sequencing (NGS) analysis was undertaken for the evaluation of the molecular basis of the disease in each family. RESULTS: A total of six children (median age 11 years) were recruited and followed prospectively for the median of 12 years. All six patients were diagnosed with LVNC by echocardiography, and five participants additionally by CMR. The presence of late gadolinium enhancement (LGE) was found in three children. Sinus bradycardia and dilation of the ascending aorta occurred in five studied patients. In four patients from three families, the molecular studies demonstrated the presence of rare heterozygous HCN4 variants. CONCLUSION: (1) The HCN4 molecular variants influence the presence of a complex LVNC phenotype, sinus bradycardia and dilation of the ascending aorta. (2) The HCN4 alteration may be associated with the early presentation of clinical symptoms and the severe course of the disease. (3) It is particularly important to assess myocardial fibrosis not only within the ventricles, but also in the atria in patients with LVNC and sinus bradycardia.

Postzygotic mosaicism of a novel PTPN11 mutation in monozygotic twins discordant for metachondromatosis.

Genetic mosaicism caused by postzygotic mutations is of a great interest due to its role in human disease. Monozygotic twins arising from a single zygote are considered as genetically identical, and any differences likely to be caused by postzygotic events. Thus, phenotypically discordant monozygotic twins offer a unique opportunity to study genotype-phenotype correlation. Here, we present a three-generation family starting from a pair of monozygotic twins discordant for metachondromatosis due to postzygotic p.(Gln175His) variant in the PTPN11 gene. Both phenotypically discordant monozygotic twins harbor p.(Gln175His), however significant differences in mosaic ratio is observed not only between twins, but also within different tissue types within one individual. Phenotypic manifestation of p.(Gln175His) in examined family clearly depends on allele variant fraction (VAF). Individuals harboring constitutional mutation (VAF 50%) present typical metachondromatosis. Milder phenotype is observed in twin harboring high-level mosaicism in the tissue of ectodermal origin (VAF 45%), but not in a blood (VAF 5%). Finally, her twin sister harboring low-level mosaicism in blood (VAF 2%) and nonblood (VAF 12%) tissues is phenotypically normal. Our results provide insights into biological role of mosaicism in disease and further support the usefulness of nonblood tissues as an optimal source of DNA for the identification of postzygotic mutations in phenotypically discordant monozygotic twins.

Prenatal Versus Postnatal Diagnosis of Meckel-Gruber and Joubert Syndrome in Patients with TMEM67 Mutations.

Renal cystic diseases are characterized by genetic and phenotypic heterogeneity. Congenital renal cysts can be classified as developmental disorders and are commonly diagnosed prenatally using ultrasonography and magnetic resonance imaging. Progress in molecular diagnostics and availability of exome sequencing procedures allows diagnosis of single-gene disorders in the prenatal period. Two patients with a prenatal diagnosis of polycystic kidney disease are presented in this article. TMEM67 mutations were identified in both fetuses using a whole-exome sequencing (WES) study. In one of them, the phenotypic syndrome diagnosed prenatally was different from that diagnosed in the postnatal period.

A recurrent de novo variant supports KCNC2 involvement in the pathogenesis of developmental and epileptic encephalopathy.

Developmental and epileptic encephalopathies (DEE) are a heterogenous group of conditions characterized by the co-occurrence of epilepsy and intellectual/developmental disability. Despite several known DEE-related genes, including these encoding ion channels, still many cases remain without molecular diagnosis. Here, we present a 2-year-old girl with severe DEE in whom whole exome sequencing revealed de novo p.(Val471Leu) variant in the KCNC2 encoding Kv3.2, a voltage-gated potassium channel. To the best of our knowledge, this is the third DEE case due to KCNC2 mutation. Our clinical and molecular findings, particularly the recurrence of p.(Val471Leu) in patient with similar clinical phenotype, further support KCNC2 as a novel DEE-associated gene.

Variable degree of mosaicism for tetrasomy 18p in phenotypically discordant monozygotic twins-Diagnostic implications.

BACKGROUND: Phenotypically discordant monozygotic twins (PDMZTs) offer a unique opportunity to study post-zygotic genetic variation and provide insights into the linkage between genotype and phenotype. We report a comprehensive analysis of a pair of PDMZTs. METHODS: Dysmorphic features and delayed neuro-motor development were observed in the proband, whereas her twin sister was phenotypically normal. Four tissues (blood, skin, hair follicles, and buccal mucosa) from both twins were studied using four complementary methods, including whole-exome sequencing, karyotyping, array CGH, and SNP array. RESULTS: In the proband, tetrasomy 18p affecting all studied tissues except for blood was identified. Karyotyping of fibroblasts revealed isochromosome 18p [i(18p)] in all metaphases. The corresponding analysis of the phenotypically normal sister surprisingly revealed low-level mosaicism (5.4%) for i(18p) in fibroblasts. CONCLUSION: We emphasize that when mosaicism is suspected, multiple tissues should be studied and we highlight the usefulness of non-invasive sampling of hair follicles and buccal mucosa as a convenient source of non-mesoderm-derived DNA, which complements the analysis of mesoderm using blood. Moreover, low-level mosaic tetrasomy 18p is well tolerated and such low-level mosaicism, readily detected by karyotyping, can be missed by other methods. Finally, mosaicism for low-level tetrasomy 18p might be more common in the general population than it is currently recognized, due to detection limitations.

The association between the insertion/deletion polymorphism of the angiotensin-converting enzyme gene and the plasma fibrinogen level in women and men with premature coronary artery atherosclerosis.

INTRODUCTION: The insertion/deletion (I/D) polymorphism of the angiotensin­converting enzyme (ACE) gene is associated with younger age at coronary artery disease (CAD) onset. Some data indicate the relationship between the DD genotype and the fibrinogen level. At the same time, the regulation of the renin-angiotensin system differs in women and men. OBJECTIVES: The objective of the study was to evaluate the sex­dependentassociation of the ACE I/D polymorphism with the plasma fibrinogen level in patients with premature CAD. PATIENTS AND METHODS: The study included 407 participants with premature CAD: 257 women not older than 55 years and 150 men not older than 45 years. Study participants had at least 1 stenosis ≥50% in a major epicardial coronary artery. The ACE I/D polymorphism (rs4343) was genotyped using polymerase chain reaction. Fibrinogen levels were measured with a modified Clauss method. We found a significant interaction indicating that sex modifies the influence of the I/D polymorphism of the ACE gene on fibrinogen levels (P = 0.02). The highest mean fibrinogen level, adjusted for age and smoking status, was observed in women with the DD genotype (575.7 mg/dl) and it was significantly higher than in men with the DD genotype (367.1 mg/dl; P <0.001) or in women with the ID genotype (491.7 mg/dl; P = 0.04). In men, there was no significant difference in mean adjusted fibrinogen levels across genotypes. CONCLUSIONS: The DD genotype of the ACE gene was associated with higher plasma fibrinogen levels in women with premature CAD yet not in men.

Analysis of De Novo Mutations in Sporadic Cardiomyopathies Emphasizes Their Clinical Relevance and Points to Novel Candidate Genes.

The vast majority of cardiomyopathies have an autosomal dominant inheritance; hence, genetic testing is typically offered to patients with a positive family history. A de novo mutation is a new germline mutation not inherited from either parent. The purpose of our study was to search for de novo mutations in patients with cardiomyopathy and no evidence of the disease in the family. Using next-generation sequencing, we analyzed cardiomyopathy genes in 12 probands. In 8 (66.7%), we found de novo variants in known cardiomyopathy genes (TTN, DSP, SCN5A, TNNC1, TPM1, CRYAB, MYH7). In the remaining probands, the analysis was extended to whole exome sequencing in a trio (proband and parents). We found de novo variants in genes that, so far, were not associated with any disease (TRIB3, SLC2A6), a possible disease-causing biallelic genotype (APOBEC gene family), and a de novo mosaic variant without strong evidence of pathogenicity (UNC45A). The high prevalence of de novo mutations emphasizes that genetic screening is also indicated in cases of sporadic cardiomyopathy. Moreover, we have identified novel cardiomyopathy candidate genes that are likely to affect immunological function and/or reaction to stress that could be especially relevant in patients with disease onset associated with infection/infestation.

Post-zygotic diploidization of triploidy in human is possible? - a case of triploid partial molar pregnancy resulting in a premature live-born diploid female infant.

OBJECTIVE: During the treatment of our patient we found that reports covering possible complications and their treatment are very scarce. Due to advancement in ultrasound diagnosis most of molar pregnancies are terminated in first trimester of pregnancy. There is the gap in knowledge concerning pregnancy complications in case of partial mole discovered in advanced pregnancy. This is why we incorporated extensive and up-to-date review of literature in our manuscript. METHOD: We described a case of previously healthy, 25 year old primigravida who delivered live daughter at 27 weeks of gestation, complicated with unusual ultrasound appearance of the placenta, severe hypotrophy, and subsequent post-partum eclampsia. RESULTS: Healthy diploid female infant, now two years old and healthy mother taking care of her. CONCLUSIONS: In clinical practice early diagnosis of this complication usually lead to pregnancy termination. In modern medicine, decisions should be based on evidence and patient-doctor mutual understanding. Termination of pregnancy with suspicion of molar placenta can be specially difficult in gestation in older nulliparous women or after ART. We sincerely hope that this report will be useful for physicians across the world in counseling and treating their patients.

Novel calcineurin A (PPP3CA) variant associated with epilepsy, constitutive enzyme activation and downregulation of protein expression.

PPP3CA encodes calmodulin-binding catalytic subunit of calcineurin, a ubiquitously expressed calcium/calmodulin-regulated protein phosphatase. Recently de novo PPP3CA variants were reported as a cause of disease in 12 subjects presenting with epileptic encephalopathy and dysmorphic features. We describe a boy with similar phenotype and severe early onset epileptic encephalopathy in whom a novel de novo c.1324C>T (p.(Gln442Ter)) PPP3CA variant was found by whole exome sequencing. Western blot experiments in patient's cells (EBV transformed lymphocytes and neuronal cells derived through reprogramming) indicate that despite normal mRNA abundance the protein expression level is strongly reduced both for the mutated and wild-type protein. By in vitro studies with recombinant protein expressed in E. coli we show that c.1324C>T (p.(Gln442Ter)) results in constitutive activation of the enzyme. Our results confirm the role of PPP3CA defects in pathogenesis of a distinct neurodevelopmental disorder including severe epilepsy and dysmorphism and provide further functional clues regarding the pathogenic mechanism.

Novel de novo mutation affecting two adjacent aminoacids in the EED gene in a patient with Weaver syndrome.

Overgrowth, macrocephaly, accelerated osseous maturation, variable intellectual disability, and characteristic facial features are the main symptoms of Weaver syndrome, a rare condition caused by mutations in EZH2 gene. Recently, in four patients with Weaver-like symptoms without mutations in EZH2 gene, pathogenic variants in EED were described. We present another patient clinically diagnosed with Weaver syndrome in whom WES revealed an EED de novo mutation affecting two neighboring aminoacids, NM_003797.3:c.917_919delinsCGG/p.(Arg306_Asn307delinsThrAsp) located in one allele (in cis). Our observation, together with previous reports suggests that EED gene testing is warranted in patients with the overgrowth syndrome features and suspicion of Weaver syndrome with normal results of EZH2 gene sequencing.

Mild Zellweger syndrome due to a novel PEX6 mutation: correlation between clinical phenotype and in silico prediction of variant pathogenicity.

Zellweger syndrome (ZS) is a consequence of a peroxisome biogenesis disorder (PBD) caused by the presence of a pathogenic mutation in one of the 13 genes from the PEX family. ZS is a severe multisystem condition characterized by neonatal appearance of symptoms and a shorter life. Here, we report a case of ZS with a mild phenotype, due to a novel PEX6 gene mutation. The patient presented subtle craniofacial dysmorphic features and slightly slower psychomotor development. At the age of 2 years, he was diagnosed with adrenal insufficiency, hypoacusis, and general deterioration. Magnetic resonance imaging showed a symmetrical hyperintense signal in the frontal and parietal white matter. Biochemical tests showed elevated liver transaminases, elevated serum very long chain fatty acids, and phytanic acid. After the death of the child at the age of 6 years, molecular diagnostics were continued in order to provide genetic counseling for his parents. Next generation sequencing (NGS) analysis with the TruSight One™ Sequencing Panel revealed a novel homozygous PEX6 p.Ala94Pro mutation. In silico prediction of variant severity suggested its possible benign effect. To conclude, in the milder phenotypes, adrenal insufficiency, hypoacusis, and leukodystrophy together seem to be pathognomonic for ZS.

Application of massively parallel sequencing (MPS) in paternity testing - case report.

Aim of the study: We present the application of massively parallel sequencing (MPS) to extend the scope of analysis in a disputed paternity case. Material and methods: A standard paternity test comprising 16 autosomal STRs was performed by capillary electrophoresis (CE) using 3130xl Genetic Analyzer. Additionally, MPS was performed with ForenSeq DNA Signature Prep Kit and Illumina MiSeq FGx™ Forensic Genomics System. Paternity index (PI) was calculated using DNAStat v.2.1 software. Results>: CE revealed two mismatches, at D21S11 and VWA, between the putative father and the child. Based on MPS results, the mismatches were analyzed and a nonconsensus sequence of allele 14 at the VWA locus in the mother - child pair was identified. Different sequence variants were also detected in 16-16 homozygote alleles at the D3S1358 locus in the child. Conclusions: MPS helped to formulate a definite conclusion regarding the paternity of the defendant and provided full information on intra-allelic polymorphism.

Haplotype dependent association of rs7927894 (11q13.5) with atopic dermatitis and chronic allergic rhinitis: A study in ECAP cohort.

The T allele of rs7927894 (at 11q13.5) was associated with atopic dermatitis and other allergic diseases. Our purpose was to replicate the association with allergic phenotypes and explore the role of rs7927894 in predisposing to persistent allergic rhinitis and atopic asthma. We also wanted to explore if other SNPs at 11q13.5 contributed to effect of rs7927894. We studied patients with atopic dermatitis (N = 270), atopic asthma (N = 486), persistent allergic rhinitis (N = 589) and controls matched for age, sex and region (N = 540, N = 372 and N = 1178, respectively). We found that rs7927894 T was associated with atopic dermatitis (OR = 1.39, CI: 1.12-1.73, P = 0.003) and independently with persistent allergic rhinitis (OR = 1.24, CI:1.07-1.43, P = 0.0043, Pcorrected = 0.013) but not atopic asthma. Analysis of additional tagging SNPs (rs7930763, rs2513517, rs7125552) showed that effect of rs7927894 T was limited to haplotypes encoding G at rs7125552. In conclusion, rs7927894 T is associated not only with atopic dermatitis but also persistent allergic rhinitis. Since these effects are haplotype dependent rs7927894 alone does not account for the association between 11q13.5 and atopic dermatitis/persistent allergic rhinitis.

OXTR polymorphism in depression and completed suicide-A study on a large population sample.

In the light of contradictory results concerning OXTR polymorphism rs53576 and depression, we decided to verify the potential association between the two on 1) a large, ethnically homogenous sample of 1185 individuals who completed the Beck Depression Inventory (BDI), as well as on 2) a sample of 763 suicide victims. In the population sample, AA males showed significantly lower BDI scores (p=0.005, pcor=0.030). Exploratory analyses suggested that this effect was limited to a subgroup within 0-9 BDI score range (p=0.0007, U-Mann Whitney test), whereas no main effect on depressive symptoms (BDI>9) was found. In the suicide sample no association with rs53576 genotype was present. Exploratory analyses in suicides revealed higher blood alcohol concentration (BAC) among AA than GG/GA males (p=0.014, U-Mann Whitney test). Our results show that the OXTR rs53576 variant modulates the mood in male individuals and may positively correlate with alcohol intake among male suicides, but is not associated with suicide or depression. The study adds to the growing knowledge on rs53576 genotype characteristics.

Further evidence for GRIN2B mutation as the cause of severe epileptic encephalopathy.

Epileptic encephalopathies (EE) include a range of severe epilepsies in which intractable seizures or severe sub-clinical epileptiform activity are accompanied by impairment of motor and cognitive functions. Mutations in several genes including ion channels and other genes whose function is not completely understood have been associated to some EE. In this report, we provide a detailed clinical description of a sporadic male patient with early-onset epilepsy and epileptic encephalopathy in whom we performed complete exome sequencing (WES) and identified a GRIN2B mutation. The GRIN2B splicing mutation in intron 10 (c.2011-1G>A) was revealed in a WES study. The result was confirmed by Sanger sequencing. No mutation was found in both parents. Our finding confirms that early-onset EE may be caused not only by gain-of-function variants but also by splice site mutations-in particular those affecting the splice acceptor site of the 10th intron of the GRIN2B gene. © 2016 Wiley Periodicals, Inc.

KIR2DS5 in the presence of HLA-C C2 protects against endometriosis.

Endometriosis is defined as the presence of functional endometrial tissue outside the uterine cavity. Several hypotheses have attempted to explain the etiology and pathogenesis of endometriosis. Recently, it has been suggested that a defect of the natural killer (NK) activity in the recognition and lysis of endometrial cells is one of the crucial points in the development of this disease. Natural killer cells can express killer immunoglobulin-like receptors (KIR), which recognize class I human leukocyte antigens on target cells. We asked whether polymorphisms in KIR, HLA-C, and HLA-B genes are risk factors for endometriosis. We tested 153 women with endometriosis diagnosed on the basis of laparoscopic and histological examination, and 213 control healthy women, who gave birth to at least one child. The frequency of KIR genes in patients was similar to that in controls except for KIR2DS5, which exerted a protective effect only in HLA-C C2-positive individuals. Moreover, KIR2DS5-positive women with endometriosis had 13 times lower chance that the disease would occupy the peritoneum than KIR2DS5- and KIR2DS4del-negative ones (OR = 0.077, P = 0.0061). Similarly, KIR2DS4del-positive endometriotic persons had 11 times lower chance for peritoneal disease (OR = 0.094, P < 0.001). Negative linkage disequilibrium between KIR2DS5 and KIR2DS4del indicates that these genes are mutually exclusive. Our data suggest that KIR2DS5 may be associated with protection from endometriosis, whereas KIR2DS4del seems to be associated with higher disease stages, possibly by exclusion of protective KIR2DS5.