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Danon disease (DD) is a rare X-linked disorder caused by pathogenic variants in LAMP2. DD primarily manifests as a severe cardiomyopathy. An early diagnosis is crucial for patient survival. The aim of the study was to determine the usefulness of ocular examination for identification of DD.Detailed ocular examination in 10 patients with DD (3 males, 7 females) and a 45-year-old asymptomatic female somatic mosaic carrier of a LAMP2 disease-causing variant.All patients with manifest cardiomyopathy had pigmentary retinopathy with altered autofluorescence and diffuse visual field loss. Best corrected visual acuity (BCVA) was decreased (0.63) in 8 (40%) out of 20 eyes. The severity of retinal pathology increased with age, resulting in marked cone-rod involvement overtime. Spectral-domain optical coherence tomography in younger patients revealed focal loss of photoreceptors, disruption and deposition at the retinal pigment epithelium/Bruch's membrane layer (corresponding to areas of marked increased autofluorescence), and hyperreflective foci in the outer nuclear layer. Cystoid macular oedema was seen in one eye. In the asymptomatic female with somatic mosaicism, the BCVA was 1.0 bilaterally. An abnormal autofluorescence pattern in the left eye was present; while full-field electroretinography was normal.Detailed ocular examination may represent a sensitive and quick screening tool for the identification of carriers of LAMP2 pathogenic variants, even in somatic mosaicism. Hence, further investigation should be undertaken in all patients with pigmentary retinal dystrophy as it may be a sign of a life-threatening disease.

Cullin 4B (CUL4B), lysosomal-associated membrane protein Type 2 (LAMP2), ATP1B4, TMEM255A, and ZBTB33 are neighboring genes on Xq24. Mutations in CUL4B result in Cabezas syndrome (CS). Male CS patients present with dysmorphic, neuropsychiatric, genitourinary, and endocrine abnormalities. Heterozygous CS females are clinically asymptomatic. LAMP2 mutations cause Danon disease (DD). Cardiomyopathy is a dominant feature of DD present in both males and heterozygous females. No monogenic phenotypes have been associated with mutations in ATP1B4, TMEM255A, and ZBTB33 genes. To facilitate diagnostics and counseling in CS and DD families, we present a female DD patient with a de novo Alu-mediated Xq24 rearrangement causing a deletion encompassing CUL4B, LAMP2, and also the other three neighboring genes. Typical to females heterozygous for CUL4B mutations, the patient was CS asymptomatic, however, presented with extremely skewed X-chromosome inactivation (XCI) ratios in peripheral white blood cells. As a result of the likely selection against CUL4B deficient clones, only minimal populations (~3%) of LAMP2 deficient leukocytes were identified by flow cytometry. On the contrary, myocardial LAMP2 protein expression suggested random XCI. We demonstrate that contiguous CUL4B and LAMP2 loss-of-function copy number variations occur and speculate that male patients carrying similar defects could present with features of both CS and DD.

X-linked adrenoleukodystrophy (X-ALD) causes cerebral adrenoleukodystrophy (cALD), myelopathy and/or adrenal insufficiency in males, and myelopathy/peripheral neuropathy in females. These distinct phenotypes are scarcely linked to a specific mutations. The objective herein was to find a link between the phenotype with the genotype mutation, serum very long-chain fatty acids (VLCFA), and the diet with Lorenzo´s and GTO oils in hemizygous males and heterozygous females.A retrospective study design with follow-up of 45 hemizygous males and 50 heterozygous females carrying mutations in ABCD1 from 35 unrelated families with X-ALD. Mutation analysis was performed by Sanger sequencing of PCR and/or RT-PCR and the severity of missense mutations was evaluated using GERP++ score and CADD score.Twenty-five described and eight novel ABCD1 mutations were identified. Fifteen males and 23 females had severe mutations while 30 males and 27 females had less detrimental ones. cALD developed in 25 males (56%) including nine boys with severe mutations, 10 boys with less detrimental mutations and 6 adults with adrenomyelopathy. Myelopathy and/or adrenal insufficiency developed in 14 males (31%), six were asymptomatic. Adrenal insufficiency developed in two of five boys treated with hematopoietic stem cell transplantation (HSCT). Myelopathy/peripheral neuropathy developed in 26% of females. No correlation was found between the disease severity and the genotype, GERP++ and CADD scores, presence/absence of aberrant ALDP protein or X-inactivation. VLCFA were higher in males than heterozygous females and decreased during Lorenzo´s and GTO oils diet without a clear clinical impact on the disease.The prognosis was unfavourable in most males and significant part of females. Therapy with early HSCT is effective. Thus, the need for early diagnosis with the neonatal screening is crucial.

Mothers with a history of gestational diabetes mellitus (GDM) have an increased risk of developing diabetes in the future and a lifelong cardiovascular risk. Postpartal expression profile of cardiovascular/cerebrovascular disease associated microRNAs was assessed 3–11 years after the delivery in whole peripheral blood of young and middle-aged mothers with a prior exposure to GDM with the aim to identify a high-risk group of mothers at risk of later development of diabetes mellitus and cardiovascular/cerebrovascular diseases who would benefit from implementation of early primary prevention strategies and long-term follow-up. The hypothesis of the assessment of cardiovascular risk in women was based on the knowledge that a series of microRNAs play a role in the pathogenesis of diabetes mellitus and cardiovascular/cerebrovascular diseases. Abnormal expression profile of multiple microRNAs was found in women with a prior exposure to GDM (miR-1-3p, miR-16-5p, miR-17-5p, miR-20a-5p, miR-20b-5p, miR-21-5p, miR-23a-3p, miR-24-3p, miR-26a-5p, miR-29a-3p, miR-100-5p, miR-103a-3p, miR-125b-5p, miR-126-3p, miR-130b-3p, miR-133a-3p, miR-143-3p, miR-145-5p, miR-146a-5p, miR-181a-5p, miR-195-5p, miR-199a-5p, miR-221-3p, miR-342-3p, miR-499a-5p, and-miR-574-3p). Postpartal combined screening of miR-1-3p, miR-16-5p, miR-17-5p, miR-20b-5p, miR-21-5p, miR-23a-3p, miR-26a-5p, miR-29a-3p, miR-103a-3p, miR-133a-3p, miR-146a-5p, miR-181a-5p, miR-195-5p, miR-199a-5p, miR-221-3p, and miR-499a-5p showed the highest accuracy for the identification of mothers with a prior exposure to GDM at a higher risk of later development of cardiovascular/cerebrovascular diseases (AUC 0.900, p < 0.001, sensitivity 77.48%, specificity 93.26%, cut off >0.611270413). It was able to identify 77.48% mothers with an increased cardiovascular risk at 10.0% FPR. Any of changes in epigenome (upregulation of miR-16-5p, miR-17-5p, miR-29a-3p, and miR-195-5p) that were induced by GDM-complicated pregnancy are long-acting and may predispose mothers affected with GDM to later development of diabetes mellitus and cardiovascular/cerebrovascular diseases. In addition, novel epigenetic changes (upregulation of serious of microRNAs) appeared in a proportion of women that were exposed to GDM throughout the postpartal life. Likewise, a previous occurrence of either GH, PE, and/or FGR, as well as a previous occurrence of GDM, is associated with the upregulation of miR-1-3p, miR-17-5p, miR-20a-5p, miR-20b-5p, miR-29a-3p, miR-100-5p, miR-125b-5p, miR-126-3p, miR-130b-3p, miR-133a-3p, miR-143-3p, miR-145-5p, miR-146a-5p, miR-181a-5p, miR-199a-5p, miR-221-3p, and miR-499a-5p. On the other hand, upregulation of miR-16-5p, miR-21-5p, miR-23a-3p, miR-24-3p, miR-26a-5p, miR-103a-3p, miR-195-5p, miR-342-3p, and miR-574-3p represents a unique feature of aberrant expression profile of women with a prior exposure to GDM. Screening of particular microRNAs may stratify a high-risk group of mothers with a history of GDM who might benefit from implementation of early primary prevention strategies.

Mucopolysaccharidosis type II (Hunter syndrome, MPS II, OMIM 309900) is an X-linked lysosomal storage disorder caused by deficiency of iduronate-2-sulfatase (IDS). We analyzed clinical and laboratory data from 44 Slavic patients with this disease. In total, 21 Czech, 7 Slovak, 9 Croatian and 7 Serbian patients (43 M/1 F) were included in the study (median age 11.0 years, range 1.2-43 years). Birth prevalence ranged from 1:69,223 (Serbia) to 1:192,626 (Czech Rep.). In the majority of patients (71%), the disease manifested in infancy. Cognitive functions were normal in 10 patients. Four, six and 24 patients had mild, moderate, and severe developmental delay, respectively, typically subsequent to developmental regression (59%). Residual enzyme activity showed no predictive value, and estimation of glycosaminoglycans (GAGs) had only limited importance for prognosis. Mutation analysis performed in 36 families led to the identification of 12 novel mutations, eight of which were small deletions/insertions. Large deletions/rearrangements and all but one small deletion/insertion led to a severe phenotype. This genotype-phenotype correlation was also identified in six cases with recurrent missense mutations. Based on patient genotype, the severity of the disease may be predicted with high probability in approximately half of MPS II patients.

Cullin 4B (CUL4B), lysosomal-associated membrane protein Type 2 (LAMP2), ATP1B4, TMEM255A, and ZBTB33 are neighboring genes on Xq24. Mutations in CUL4B result in Cabezas syndrome (CS). Male CS patients present with dysmorphic, neuropsychiatric, genitourinary, and endocrine abnormalities. Heterozygous CS females are clinically asymptomatic. LAMP2 mutations cause Danon disease (DD). Cardiomyopathy is a dominant feature of DD present in both males and heterozygous females. No monogenic phenotypes have been associated with mutations in ATP1B4, TMEM255A, and ZBTB33 genes. To facilitate diagnostics and counseling in CS and DD families, we present a female DD patient with a de novo Alu-mediated Xq24 rearrangement causing a deletion encompassing CUL4B, LAMP2, and also the other three neighboring genes. Typical to females heterozygous for CUL4B mutations, the patient was CS asymptomatic, however, presented with extremely skewed X-chromosome inactivation (XCI) ratios in peripheral white blood cells. As a result of the likely selection against CUL4B deficient clones, only minimal populations (~3%) of LAMP2 deficient leukocytes were identified by flow cytometry. On the contrary, myocardial LAMP2 protein expression suggested random XCI. We demonstrate that contiguous CUL4B and LAMP2 loss-of-function copy number variations occur and speculate that male patients carrying similar defects could present with features of both CS and DD.

The aim of the study was to verify if quantification of placental specific C19MC microRNAs in plasma exosomes would be able to differentiate during the early stages of gestation between patients subsequently developing pregnancy-related complications and women with the normal course of gestation and if this differentiation would lead to the improvement of the diagnostical potential. The retrospective study on singleton Caucasian pregnancies was performed within 6/2011-2/2019. The case control study, nested in a cohort, involved women that later developed GH (n = 57), PE (n = 43), FGR (n = 63), and 102 controls. Maternal plasma exosome profiling was performed with the selection of C19MC microRNAs with diagnostical potential only (miR-516b-5p, miR-517-5p, miR-518b, miR-520a-5p, miR-520h, and miR-525-5p) using real-time RT-PCR. The down-regulation of miR-517-5p, miR-520a-5p, and miR-525-5p was observed in patients with later occurrence of GH and PE. Maternal plasma exosomal profiling of selected C19MC microRNAs also revealed a novel down-regulated biomarker during the first trimester of gestation (miR-520a-5p) for women destinated to develop FGR. First trimester circulating plasma exosomes possess the identical C19MC microRNA expression profile as placental tissues derived from patients with GH, PE and FGR after labor. The predictive accuracy of first trimester C19MC microRNA screening (miR-517-5p, miR-520a-5p, and miR-525-5p) for the diagnosis of GH and PE was significantly higher in the case of expression profiling of maternal plasma exosomes compared to expression profiling of the whole maternal plasma samples.

Acetyl-CoA:α-glucosaminide N -acetyltransferase ( N -acetyltransferase) is a lysosomal membrane enzyme that catalyzes a key step in the lysosomal degradation of heparan sulfate. Its deficiency causes Sanfilippo syndrome type IIIC (Mucopolysaccharidosis type IIIC, MPS IIIC). Here we characterize the promoter region of HGSNAT , the gene encoding N -acetyltransferase, which is located in the pericentromeric region of chromosome 8. We show that HGSNAT transcription is driven by a TATA-less promoter whose key elements are contained within the 1054 bp region upstream of exon 1. About 400 bases of the region's 3′-prime end overlap with an unmethylated CpG island. Reduced reporter activities from promoter serial deletion constructs suggested strong regulatory elements at positions − 101 to − 20 bp and − 1073 to − 716 bp of the downstream initiation codon (DS-ATG). Targeted mutagenesis of the first Specificity protein 1-A (Sp1-A) of the six in silico-predicted Sp1 sites in the region flanking the major transcription start sites (TSSs, + 50/− 101) led to a 55% decrease of reporter activity, while inactivation of each of Sp1-B and Sp1-C resulted in its almost two-fold increase. The binding of Sp1 to the region was confirmed by chromatin immunoprecipitation (ChIP). Overall, this confirms that Sp1 is important for regulation of the HGSNAT promoter. Promoter fragments in antisense orientation (constructs pGL4 − 20/− 1305 and pGL4 + 50/− 1305) led to reporter activities of about 50% of the pGL4 − 1305/− 20 activity, implying divergent initiation of transcription at the promoter. We identified two main TSSs at positions + 1 and − 15 from DS-ATG using Rapid amplification of cDNA ends (5′RACE). Transcripts initiating at the TSSs thus contain only DS-ATG. Five patients from our MPS IIIC cohort (n = 23) carried the rs4523300 promoter variant and one the rs149596192 promoter variant. Both variants lowered the expression of the reporter down to 68% and 59%, respectively. However, white blood cell (WBC) N -acetyltransferase activities in individuals carrying the variants did not significantly differ from homozygotes for the wild-type alleles, suggesting only a partial impact of transcriptional regulation on N -acetyltransferase activities in vivo.

Background and methods Gestational hypertension (GH), preeclampsia (PE) and fetal growth restriction (FGR) may predispose to later onset of cardiovascular/cerebrovascular diseases. We examined if pregnancy complications induce postpartum alterations in gene expression of cardiovascular/cerebrovascular disease associated microRNAs. 29 microRNAs were tested in peripheral blood of women, compared between groups with a history of GH, PE, FGR and controls, and correlated with the severity of the disease regarding clinical signs, delivery date, and Doppler parameters. Results GH was associated with the up-regulation of miR-20a-5p, miR-143-3p, miR-146a-5p, miR-181a-5p, miR-199a-5p, miR-221-3p, and miR-499a-5p. The up-regulation of miR-17-5p, miR-20b-5p, miR-29a-3p, and miR-126-3p was a mutual phenomenon of GH and severe PE. GH and early PE were associated with up-regulation of miR-1-3p and miR-17-5p. GH and late PE showed up-regulation of miR-17-5p, miR-20b-5p, and miR-29a-3p. Severe PE induced up-regulation of miR-133a-3p and down-regulation of miR-130b-3p. MiR-133a-3p up-regulation was also observed in early PE. PE and/or FGR with abnormal Doppler parameters demonstrated up-regulation of miR-100-5p, miR-125b-5p, miR-133a-3p, and miR-145-5p. The combination screening was superior over using individual microRNAs for patients with GH, PE regardless of the severity of the disease, severe PE and early PE. A cardiovascular risk at patients with late PE, PE and/or FGR with abnormal Doppler parameters was identified more accurately using the single microRNA only. Conclusion Epigenetic changes characteristic for cardiovascular/cerebrovascular diseases are present in women with a prior exposure to pregnancy complications. Screening of microRNAs may be used to identify patients at a higher risk of later development of cardiovascular/cerebrovascular diseases.

Danon disease (DD) is an X-linked disorder caused by mutations in the lysosomal-associated membrane protein 2 (LAMP2) gene (Xq24). DD is characterized by cognitive deficit, myopathy, and cardiomyopathy in male patients. The phenotype is variable and mitigated in females. The timely identification of de-novo LAMP2 mutated family members, many of whom are heterozygous females, remains critical for their treatment and family counseling. DD laboratory testing builds on minimally invasive quantification of the LAMP2 protein in white blood cells and characterization of the specific mutation. This integrative approach is particularly helpful when assessing suspect female heterozygotes. LAMP2 exon-copy number variations (eCNVs) were so far reported only in X-hemizygous male DD probands. In heterozygous female DD probands, the wild-type allele may hamper the identification of an eCNV even if it results in the complete abolition of LAMP2 transcription and/or translation. To document the likely underappreciated rate of occurrence and point out numerous potential pitfalls of detection of the LAMP2 eCNVs, we present the first two DD heterozygote female probands who harbor novel multi-exon LAMP2 deletions. Critical for counseling and recurrence prediction, we also highlight the need to search for somatic-germinal mosaicism in DD families.

The HUMARA assay, the most common method for evaluation of X-inactivation skewing in blood cells, has been reported to be usable in only about 80% of females, emphasizing the need for alternative methods for testing of HUMARA-uninformative individuals. We conducted an in silico search for potentially polymorphic tri-to-hexanucleotide repeats in the proximity of CpG islands located in 5' regions of X-chromosome genes to design five candidate assays (numbered I, II, III, IV, and V) combining methylation-specific restriction digest with PCR amplification in a manner similar to the HUMARA assay. The results obtained by these assays in 100 healthy females were compared to X-inactivation skewing measured by the AR-MSP method which is based on methylation-specific PCR amplification of the first exon of the AR gene. On the basis of statistical evidence, three of the novel assays (II, IV, and V), which were informative in 18%, 61%, and 55% of females in the cohort, respectively, may be used as alternatives or conjointly with the HUMARA assay to improve its reliability. The three new assays were combined with the HUMARA assay into a novel X-inactivation test leading to the increase of informative females in the cohort from 67% to 96%.

Ornithine transcarbamylase (OTC) deficiency is an X-linked disorder that causes recurrent and life-threatening episodes of hyperammonemia. The clinical picture in heterozygous females is highly diverse and derives from the genotype and the degree of inactivation of the mutated X chromosome in hepatocytes. Here, we describe molecular genetic, biochemical, and histopathological findings in the livers explanted from two female patients with late-onset OTC deficiency. Analysis of X-inactivation ratios by DNA methylation-based assays showed remarkable intra-organ variation ranging from 46:54 to 82:18 (average 70:30, n = 37), in favor of the active X chromosome carrying the mutation c.583G>C (p.G195R), in the first patient and from 75:25 to 90:10 (average 82:18, n = 20) in favor of the active X chromosome carrying the splicing mutation c.663+1G>A in the second patient. The X-inactivation ratios in liver samples correlated highly with the proportions of OTC-positive hepatocytes calculated from high-resolution image analyses of the immunohistochemically detected OTC in frozen sections that was performed on total area > 5 cm2. X-inactivation ratios in blood in both female patients corresponded to the lower limit of the liver values. Our data indicate that the proportion of about 20–30% of hepatocytes expressing the functional OTC protein is not sufficient to maintain metabolic stability. X-inactivation ratios assessed in liver biopsies taken from heterozygous females with X-linked disorders should not be considered representative of the whole liver.

Alu-mediated tandem duplication of exons 4 and 5 (g.15815_22218dup6404) is a novel mutation that has been detected in the LAMP2 gene (Xq24). This exon copy number variation was found in two brothers with the typical phenotype of Danon disease, including characteristic myocardial changes on magnetic resonance imaging. The 6.4 kb duplication was identified in both boys by a combination of exon dosage qPCR analyses and duplication breakpoint/junction mapping. The rearrangement results in a plethora of abnormal LAMP2 splicing variants and also in use of likely cryptic splice sites in the 3′ terminus of LAMP2 gene. Although we found minute amounts of normal LAMP2B and LAMP2A mRNAs, no protein was detectable in peripheral blood leukocytes by flow cytometry in both brothers. Uniquely, the fraction of LAMP2-deficient granulocytes (0.06 %) assessed by flow cytometry in the patients’ asymptomatic mother substantially differed from the random distribution of X-chromosome inactivation in her leukocytes. This discrepancy was later explained by molecular genetic methods as a consequence of mosaic distribution of the mutation in her somatic tissues. Altogether, we report a novel and mosaically distributed exon copy number rearrangement in the LAMP2 gene and comment on obstacles this genetic setup presents to the overall cellular and molecular diagnostic algorithm of Danon disease. Our observations of the mosaicism in the asymptomatic mother suggest that similarly affected females could be a potentially under-diagnosed Danon disease carrier group and that LAMP2 flow cytometry, because of its supreme sensitivity, can be an efficient method for pedigree screening.

Ornithine carbamoyltransferase deficiency is the most common inherited defect of the urea cycle. We examined 28 male and 9 female patients from 29 families and identified 25 distinct mutations in OTC, 14 of which were novel. Three novel missense mutations (p.Ala102Pro, p.Pro158Ser, p.Lys210Glu) and a novel deletion of the Leu43 are not directly involved either in the enzyme active site or in the intersubunit interactions; however, the mutations include conserved residues involved in intramolecular interaction network essential for the function of the enzyme. Three novel large deletions - a 444 kb deletion affecting RPGR, OTC and TSPAN7, a 10 kb-deletion encompassing OTC exons 5 and 6 and a 24.5 kb-deletion encompassing OTC exons 9 and 10 - have probably been initiated by double strand breaks at recombination-promoting motifs with subsequent non-homologous end joining repair. Finally, we present a manifesting heterozygote carrying a hypomorphic mutation p.Arg129His in combination with unfavorably skewed X-inactivation in three peripheral tissues.

Background & objectives: Heat shock proteins (Hsp) are ubiquitously distributed phylogenetically conserved molecules that regulate cellular homeostasis and maintain the integrity and function of cellular proteins. Increased levels of Hsp in maternal circulation have been shown to be associated with increased risk of pregnancy related complications. The objective of this study was to explore extracellular Hsp mRNA levels in maternal circulation and quantified Hsp27, Hsp60, Hsp70, Hsp90 and Hsp70 binding protein 1 (HspBP1) mRNAs in maternal plasma samples using real-time reverse-transcriptase polymerase chain reaction. Methods: Pregnancies with gestational hypertension (GH) (n = 33), pre-eclampsia (PE) with or without foetal growth restriction (FGR) (n = 78) and FGR (n = 25) were involved in the study. Hsp gene expression was analysed in relation to the severity of the disease with respect to the degree of clinical signs, requirements for the delivery and Doppler ultrasound parameters. Results: Upregulation of Hsp70 was observed in patients with mild and severe PE (P = 0.004 and P = 0.005, respectively) and in pregnancies complicated with PE delivering before and after 34 wk of gestation regardless of the degree of clinical signs (P = 0.015 and P = 0.009, respectively). No difference in the expression of other Hsp genes among the studied groups was observed. No association between Hsp gene expression and Doppler ultrasonography parameters was found. Interpretation & conclusions: These data support that maternal circulation can reflect both maternal and foetal pathologic conditions. Hsp70 represents the sole plasmatic marker, and increased Hsp70 mRNA levels reflect maternal and placental stress response to pregnancy-related complications such as GH and PE, irrespective of the severity of the disease.

PROBLEM Investigating the stress response in the central cotyledon zone of placental tissue in pregnancies with PPROM, PTB, and at term in labor. METHOD OF STUDY Gene expression of Hsp27, Hsp60, Hsp70, Hsp90, and HspBP1 was compared between these particular groups. Correlation between variables including Hsp gene expression in placental tissue and the gestational age at delivery, WBC count at admission, and serum levels of CRP at admission in patients with PPROM and PTB was determined. RESULTS Both PPROM and PTB pregnancies were associated with altered Hsp gene expression profile. While PPROM and PTB always induced upregulation of Hsp27 and Hsp60, downregulation of Hsp70 and HspBP1 was present entirely in patients with PPROM. HspBP1 expression profile was also able to differentiate between PPROM and PTB pregnancies. The highest mRNA levels of Hsp60 and Hsp70 were detected in PTB pregnancies with elevated CRP levels at admission. Some of the examined Hsp displayed increased expression with advancing gestational age in both groups (PPROM: Hsp27, Hsp70, and Hsp90; and PTB: Hsp27). CONCLUSION Upregulation of Hsp27 is a common phenomenon shared between pregnancies affected with PTB and PPROM. On the other hand, downregulation of Hsp70 and HspBP1 represents a unique feature of PPROM.

Cataplexy is the sudden muscle weakness brought on by strong emotions, particularly joking, laughter, or anger. Cataplexy may involve only certain group of muscles or the entire voluntary musculature. In rare cases, symptoms of cataplexy can be seen during the course of some inherited diseases (Niemann-Pick type C (NPC), Prader-Willi syndrome, myotonic dystrophy, Norrie disease). We report the successful use of miglustat, a reversible inhibitor of the enzyme glucosylceramide synthase, approved for use in Gaucher's disease, and which catalyses the first step in the biosynthesis of most glycosphingolipid, in a boy with NPC with cataplexy. A 9-year-old boy was admitted for assessments of frequent "drop attacks" while laughing. The filipin fluorescence tests of cultured skin fibroblasts revealed massive accumulation of unesterified cholesterol, confirming the diagnosis of NPC disease. Molecular studies confirmed the diagnosis of NPC too. After approval from the bioethics committee, miglustat was initiated on the child at 100mg three times a day. Cataplectic attacks disappeared completely after 6 months on treatment, and patient continues to be in remission from the cataplectic attacks at 16 months follow-up. There was no further progression of neurological signs or symptoms or splenomegaly, with some improvement in cognitive function as well as social, affective and attention problems, up-gaze, and gait. Miglustat was well tolerated with no side effects observed. In summary, this is the first report of miglustat treatment of cataplexy in NPC. Long-term follow-up for continuing efficacy and tolerability in a larger cohort with NPC is needed to substantiate our observation.

In a female patient with signs of ornithine carbamoyltransferase deficiency (OTCD), the only variation found was a heterozygous single nucleotide substitution c.-366A>G. Determination of transcription start sites of human OTC 95, 119 and 169 bp upstream of the initiation codon located the variation upstream of the 5'-untranslated region. We predicted the human promoter and enhancer elements from homology with rat and mouse, performed function analysis of both regulatory regions and assessed the impact of the promoter variation in functional studies using dual luciferase reporter assay. Our data indicate that: (i) Full transcriptional activity of human OTC promoter depends on an upstream enhancer, as do the rodent promoters. (ii) The promoter variation c.-366A>G does not affect the function of the promoter alone but it disrupts the interaction of the promoter with the enhancer. (iii) The promoter-enhancer interaction contributes to tissue specific expression of OTC in the liver. We conclude that mutations in the regulatory regions of OTC can lead to OTCD and should be included in genetic testing.

Mucopolysaccharidosis type I (MPS I) is an autosomal recessive lysosomal storage disorder that is caused by a deficiency of the enzyme α-l-iduronidase (IDUA). Of the 21 Czech and Slovak patients who have been diagnosed with MPS I in the last 30 years, 16 have a severe clinical presentation (Hurler syndrome), 2 less severe manifestations (Scheie syndrome), and 3 an intermediate severity (Hurler/Scheie phenotype). Mutation analysis was performed in 20 MPS I patients and 39 mutant alleles were identified. There was a high prevalence of the null mutations p.W402X (12 alleles) and p.Q70X (7 alleles) in this cohort. Four of the 13 different mutations were novel: p.V620F (3 alleles), p.W626X (1 allele), c.1727 + 2T > G (1 allele) and c.1918_1927del (2 alleles). The pathogenicity of the novel mutations was verified by transient expression studies in Chinese hamster ovary cells. Seven haplotypes were observed in the patient alleles using 13 intragenic polymorphisms. One of the two haplotypes associated with the mutation p.Q70X was not found in any of the controls. Haplotype analysis showed, that mutations p.Q70X, p.V620F, and p.D315Y probably have more than one ancestor. Missense mutations localized predominantly in the hydrophobic core of the enzyme are associated with the severe phenotype, whereas missense mutations localized to the surface of the enzyme are usually associated with the attenuated phenotypes. Mutations in the 130 C-terminal amino acids lead to clinical manifestations, which indicates a functional importance of the C-terminus of the IDUA protein. © 2009 Wiley-Liss, Inc.

WOS: 000264889000024, PubMed ID: 19201763, The neuronal ceroid lipofuscinoses (NCLs), the most common neurodegenerative disorders of childhood, are characterized by the accumulation of autofluorescent storage material mainly in neurons. Although clinically rather uniform, variant late-infantile onset NCL (vLINCL) is genetically heterogeneous with four major underlying genes identified so far. We evaluated the genetic background underlying vLINCL in 119 patients, and specifically analysed the recently reported CLN7/MFSD8 gene for mutations in 80 patients. Clinical data were collected from the CLN7/MFSD8 mutation positive patients. Eight novel CLN7/MFSD8 mutations and seven novel mutations in the CLN1/PPT1, CLN2/TPP1, CLN5, CLN6 and CLN8 genes were identified in patients of various ethnic origins. A significant group of Roma patients originating from the former Czechoslovakia was shown to bear the c.881CA (p.Thr294Lys) mutation in CLN7/MFSD8, possibly due to a founder effect. With one exception, the CLN7/MFSD8 mutation positive patients present a phenotype indistinguishable from the other vLINCL forms. In one patient with an in-frame amino acid substitution mutation in CLN7/MFSD8, the disease onset was later and the disease course less aggressive than in variant late-infantile NCL. Our findings raise the total number of CLN7/MFSD8 mutations to 14 with the majority of families having private mutations. Our study confirms that CLN7/MFSD8 defects are not restricted to the Turkish population, as initially anticipated, but are a relatively common cause of NCL in different populations. CLN7/MFSD8 should be considered a diagnostic alternative not only in variant late-infantile but also later onset NCL forms with a more protracted disease course. A significant number of NCL patients in Turkey exist, in which the underlying genetic defect remains to be determined., Center of Excellence in Complex Disease Genetics of the Academy of FinlandAcademy of Finland; Folkhalsan Research Foundation; Institute of Inherited Metabolic Disorders support [MSM 0021620806]; Ministry of Health of the Czech Republic grantMinistry of Health, Czech Republic [NR/8351-3]; Wellcome TrustWellcome Trust [054606]; Batten Disease Support and Research Association; Helsinki Biomedical Graduate School, We thank the families that participated in this study, as well as Marianne Rohrbach, Stacey Hewson, Eva Kostalova, Gul Serdaroglu, Larisa Stolnaja, Claudia Kitzmuller, Ahmed Mohamed and Teija-Tuulia Toivonen for their contribution. This study was supported by the Center of Excellence in Complex Disease Genetics of the Academy of Finland, the Folkhalsan Research Foundation, the Institute of Inherited Metabolic Disorders support (project MSM 0021620806), the Ministry of Health of the Czech Republic grant (NR/8351-3), the Wellcome Trust (054606) and the Batten Disease Support and Research Association. M. K. is fellow of the Helsinki Biomedical Graduate School. B. A. M. holds a Canada Research Chair in Pediatric Neurogenetics.

Female carriers of hypoxanthine-guanine phosphoribosyltransferase (HPRT) deficiency have somatic cell mosaicism of HPRT activity and are healthy. We report a 50-year-old woman without gout or nephrolithiasis. She was never on allopurinol. Normal serum uric acid concentrations, increased plasma hypoxanthine, and xanthine were found. HPRT activity in erythrocytes was surprisingly low: at 8.6 nmol h(-1) mg (-1) haemoglobin. Mutation analysis revealed a heterozygous HPRT gene mutation, c.215A > G (p.Tyr72Cys). Assessment of X-inactivation ratio has shown that > 75% of the active X-chromosome bears the mutant allele and could explain these unusual, previously undescribed findings.

We have identified 21 different alpha-galactosidase A gene (GLA) mutations in 22 unrelated Czech and Slovak families with Fabry disease. Eleven of these mutations were novel (point mutations D93N, A135V, D155H, G171R, Q280K, G360S, Q330X, splicing errors c.194ins14, c.801ins36 and deletions c.674_732del59, g.3405_6021del2617). Genotyping of family members for family-specific mutations revealed 55 heterozygotes that manifested clinical symptoms of different severity. To examine the contribution of X-inactivation skewing to disease manifestation in Fabry heterozygotes, we have adopted the Mainz severity scoring scheme and compared the score values with the X-inactivation status in 39 carriers in an age-dependent manner. The age-score trendline of Fabry females who had a predominantly inactivated X-chromosome bearing a wild-type GLA allele (10 of 38 females) was markedly steeper than in the rest of the cohort. One female carrier with an inactivated mutated allele had a low score value when compared to the other heterozygotes of the same age. These data suggest that X-inactivation is indeed a major factor determining the severity of clinical involvement in Fabry heterozygotes. There was a statistically significant difference between the severity score values of heterozygotes with random and non-random X-chromosome inactivation at the 5% level of significance. Further studies will show if the degree of the wildtype allele inactivation will be useful as a predictive marker of severity of phenotype in Fabry heterozygotes. Although the correlation between X-inactivation skewing and presentation of the disease in Fabry heterozygotes has previously been suggested in the literature, this report is among the first attempts to examine this relationship systematically.

Ornithine transcarbamylase (OTC) deficiency, transmitted as an X-linked trait, is the most common disorder of the urea cycle. At least 3.5% out of more than 230 mutations consist of large gene deletions, involving one or more exons. Only in 78% of OTC patients the diagnosis was confirmed on DNA level. We analysed OTC intragenic polymorphisms and haplotypes, in an attempt to contribute to the clarification of unresolved cases, in three populations (Czech, Portuguese, and Mozambican) and identified six novel nucleotide changes, all of them occurring with frequency higher than 12.5% in Europeans. Five of these polymorphisms occur with a significant frequency also in Africans. The number and frequency of haplotypes defined with the newly reported markers differ in individual populations.

The study describes the stress response in the central cotyledon zone of placental tissue and in maternal whole peripheral blood to pregnancy related complications including gestational hypertension (n = 31), preeclampsia w or w/o fetal growth restriction (n = 95), and fetal growth restriction (n = 39) using real-time RT-PCR and genes encoding Hsp27, Hsp60, Hsp70, Hsp90 and HspBP1 proteins. The placental tissue does not respond to pregnancy induced hypertension, fetal growth restriction and short-term severe preeclampsia that requires immediate termination of gestation. Upregulation of Hsp27, Hsp90 and HspBP1 appears just in case of long-term deteriorated conditions (usually in mild preeclampsia, that enable further continuation of gestation, when properly treated). On the other hand, maternal circulation is able to reflect both maternal and fetal pathologic conditions. While pregnancy related complications always induce upregulation of Hsp70 and downregulation of Hsp90 in maternal whole peripheral blood, the increase of Hsp60 mRNA levels occurs entirely in patients with preeclampsia and/or fetal growth restriction. Hsp60, Hsp70 and Hsp90 are dysregulated in maternal circulation irrespective of the severity of the disease (in both mild and severe preeclampsia) and the requirements for the delivery (before and after 34th week of gestation). Nevertheless, the highest Hsp60 mRNA levels may be observed in pregnancies with signs of the centralization of the fetal circulation associated with fetal hypoxia.

Benign recurrent intrahepatic cholestasis (BRIC) is a rare inherited liver disease characterized by recurrent attacks of severe cholestasis with no progression to end stage liver disease. Patients have jaundice, however, serum gamma-glutamyltransferase and cholesterol levels remain within the normal range during the attacks. Three mutations in the familial intrahepatic cholestasis 1 (ATP8B1) gene encoding a P-type ATPase have been reported so far in patients with the autosomal recessive form of BRIC. A novel rare type insertion-deletion mutation, also called indel, was found in exon 24 of ATP8B1 in our patient together with a known missense mutation 1982T>C in exon 17. The mechanism of the indel formation is proposed and impact of the indel mutation on the function of ATP8B1 protein is discussed.

X-linked adrenoleukodystrophy (X-ALD) is an inherited metabolic disease associated with mutations in the ABCD1 gene that encodes an ATP-binding cassette transporter protein, ALDP. The disease is characterized by increased concentrations of very long-chain fatty acids (VLCFAs) in plasma and in adrenal, testicular and nervous tissues, due to a defect in peroxisomal VLCFA beta-oxidation. In the present study, we analyzed 10 male patients and 17 female carriers from 10 unrelated pedigrees with X-ALD from Argentina. By sequencing the ABCD1 we detected 9 different mutations, 8 of which were novel. These new mutations were verified by a combination of methods that included both functional (western blot and peroxisomal VLCFA beta-oxidation) and bioinformatics analysis. The spectrum of novel mutations consists of 3 frameshift (p.Ser284fs*16, p.Glu380Argfs*21 and p.Thr254Argfs*82); a deletion (p.Ser572_Asp575del); a splicing mutation (c.1081+5G>C) and 3 missense mutations (p.Ala341Asp, p.His420Pro and p.Tyr547Cys). In one patient 2 changes were found: a known missense (p.His669Arg) and an unpublished amino acid substitution (p.Ala19Ser). In vitro studies suggest that p.Ala19Ser is a polymorphism. Moreover, we identified two novel intronic polymorphisms and two amino acid polymorphisms. In conclusion, this study extends the spectrum of mutation in X-ALD and facilitates the identification of heterozygous females

Danon disease (DD) is a monogenic X-linked disorder characterized by cardiomyopathy, skeletal myopathy and variable degrees of intellectual disability. DD develops due to mutations in the gene encoding lysosomal-associated membrane protein 2 (LAMP2). We report on a family exhibiting the clinical phenotype comprising of hypertrophic cardiomyopathy and ventricular pre-excitation, myopia and mild myopathy in two male patients and cardiomyopathy and myopia in a female patient. The diagnosis of DD in this family was based on the assessment of the clinical phenotypes and the absence of LAMP2 in skeletal and/or cardiac muscle biopsy specimens. Sequence analysis of the LAMP2 gene and its mRNA revealed a novel LAMP2 mutation (c.940delG) in all three patients. Approximately 25% of the female patient's cardiomyocytes were LAMP2 positive apparently due to the unfavorable skewing of X chromosome inactivation. We further performed qualitative LAMP2 immunohistochemistry on peripheral white blood cells using the smear technique and revealed the absence of LAMP2 in the male patients. LAMP2 expression was further assessed in granulocytes, CD4+ and CD8+ T lymphocytes, CD20+ B lymphocytes, CD14+ monocytes and CD56+ natural killer cells by quantitative polychromatic flow cytometry. Whereas the male DD patients lacked LAMP2 in all WBC populations, the female patient expressed LAMP2 in 15.1% and 12.8% of monocytes and granulocytes, respectively. LAMP2 expression ratiometrics of highly vs. weakly expressing WBC populations discriminated the DD patients from the healthy controls. WBCs are thus suitable for initial LAMP2 expression testing when DD is a differential diagnostic option. Moreover, flow cytometry represents a quantitative method to assess the skewing of LAMP2 expression in female heterozygotes. Because LAMP2 is a major protein constituent of the membranes of a number of lysosome-related organelles, we also tested the exocytic capacity of the lytic granules from CD8+ T lymphocytes in the patient samples. The degranulation triggered by a specific stimulus (anti-CD3 antibody) was normal. Therefore, this process can be considered LAMP2 independent in human T cells. The c.940delG mutation results in a putatively truncated protein (p.A314QfsX32), which lacks the transmembrane domain and the cytosolic tail of the wild-type LAMP2. We tested whether this variant becomes exocytosed because of a failure in targeting to late endosomes/lysosomes. Western blotting of cardiac muscle, WBCs and cultured skin fibroblasts (and their culture media) showed no intra- or extracellular truncated LAMP2. By comparing the expression pattern and intracellular targeting in cultured skin fibroblasts of normal LAMP2 isoforms (A, B and C) tagged with green fluorescent protein (GFP) and the A314Qfs32-GFP fusion, we found that the A314Qfs32-GFP protein is not even expressed. These observations suggest that the truncated protein is unstable and is co-translationally or early post-translationally degraded.

Skin penetration enhancers are compounds used to facilitate the transdermal delivery of drugs that are otherwise not sufficiently permeable. Through a synthetic route implementing two series of esters, we generated transdermal penetration enhancers by a multi-step reaction with substituted 6-aminohexanoic acid. We present the synthesis of all newly prepared compounds here with structural confirmation accomplished by 1 H NMR, 13 C NMR, IR and mass spectroscopy (MS). The lipophilicity (log k ) of all compounds was determined via RP-HPLC and their hydrophobicity (log P / C log P ) was also calculated using two commercially available programs. Ab initio calculations of geometry and molecular dynamic simulations were employed to investigate the 3-dimensional structures of selected compounds. The transdermal penetration-enhancing activity of all the synthesized esters were examined in vitro and demonstrated higher enhancement ratios than oleic acid. Compounds 2e (C 10 ester chain) and 2f (C 11 ester chain) exhibited the highest enhancement ratios. It can be concluded that the series non-substituted at the C (2) position by a ω-lactam ring showed significantly higher activity than those with azepan-2-one. None of the prepared compounds penetrated through the skin. All of the investigated agents demonstrated minimal anti-proliferative activity using the SK-N-MC neuroepithelioma cell line (IC 50 >6.25 μM), suggesting these analogs would have a low cytotoxic profile when administered in vivo as chemical penetration enhancers. The correlation between the chemical structure of the studied compounds and their lipophilicity is discussed in regards to transdermal penetration-enhancing activity.

Skin penetration enhancers are used in the formulation of transdermal delivery systems for drugs that are otherwise not sufficiently skin-permeable. We generated two series of esters by multi-step synthesis with substituted 6-aminohexanoic acid as potential transdermal penetration enhancers by multi-step synthesis. The synthesis of all newly prepared compounds is presented here. Structure confirmation of all generated compounds was accomplished by 1 H NMR, 13 C NMR, IR and MS spectroscopy. All the prepared compounds were analyzed using RP-HPLC and their lipophilicity (log k ) was determined. The hydrophobicity (log P / C log P ) of the studied compounds was also calculated using two commercially available programs and 3D structures of the selected compounds were investigated by means of ab initio calculations of geometry and molecular dynamic simulations. All the synthesized esters were tested for their in vitro transdermal penetration-enhancing activity and showed higher enhancement ratios than oleic acid. The highest enhancement ratios were exhibited by compound 5f (C (2) substituted with piperidine-2-one, C 11 ester chain) and 5a (C (2) substituted with piperidine-2-one, C 6 ester chain). The series with a ω-lactam ring (piperidin-2-one; 5a – g ), showed slightly higher activities than those with morpholine ( 6a – 6g ). All of the agents showed minimal anti-proliferative activity (IC 50 >6.25 μM), indicating they would have low cytotoxicity when administered as chemical penetration enhancers. The relationships between the lipophilicity and the chemical structure of the studied compounds, as well as the correlation between their chemical structure and transdermal penetration-enhancing activity, are discussed.

We describe a girl with mild facial anomalies, mild mental retardation, and atypical autism with a remarkable behavioral phenotype of persistent anger, aggression, and dysphoria. The occurrence of late-onset tremor and premature ovarian failure in the maternal branch of the family pointed to a possible defect in the FMR1 gene. Indeed, the patient carried a full FMR1 mutation. Unexpectedly, both alleles of the gene were almost completely methylated. Cytogenetic examination of the patient revealed in addition a large de novo deletion in band Xp22 on one of her X chromosomes. The deletion was fine mapped using oligonucleotide array CGH, and its breakpoints were localized using sequencing. The size of the deletion was about 17.4 Mb, and it contained more than 90 protein-coding genes. Microsatellite analysis indicated paternal origin of the aberrant chromosome. The large rearrangement was the most probable cause of the X-inactivation skewing, thus explaining the methylation of not only the expanded (maternal) but also the normal (paternal) FMR1 alleles. This pattern of skewed X-inactivation was confirmed using the analysis of methylation at the AR locus. The relatively mild phenotype of the patient resulted most likely from unmasking of the FMR1 defect. Although the deleted region contained many important genes, the phenotypic contribution of the rearranged X chromosome was probably limited by its almost complete inactivation. However, reduced dose of several genes escaping X-inactivation might also play a role in the phenotype of the patient. © 2010 Wiley-Liss, Inc.

Background Mutations in the α- l -iduronidase A (IDUA) gene cause mucopolysaccharidosis type I (MPS I), a progressive multisystem disorder with features ranging over a continuum from mild to severe which is inherited in an autosomal recessive manner. To date over 100 mutations are known, nonetheless genotype–phenotype prediction is complicated and hampered due to attenuating polymorphisms, rare sequence variants, varied genetic backgrounds and environmental effects. Methods In this study we report the first development of a denaturating high performance liquid chromatography (dHPLC) protocol for the rapid and accurate detection of recently described mutations in the IDUA gene. Optimal PCR running and dHPLC partial denaturing conditions for mutation detection were established for each PCR amplicon corresponding to 14 IDUA exons and their adjacent intronic/flanking sequences. Results A total of 12 different mutations, 5 nonsense, 4 missense, 1 deletion, and 2 splice site (intron), in 10 MPS I patients were screened. All mutations revealed a distinct dHPLC pattern thus enabling their accurate detection. Conclusions A dHPLC screening method was developed for the detection of mutations and sequence variants in the IDUA gene and the results presented in this study revealed that this promising method proved to be robust, automated, economical and above all, highly sensitive. Costs for the detection of mutations causing MPS I disease should be reduced by using this method as a pre-analytical tool followed by sequencing of aberrant heteroduplex-forming amplicons.

The aim of this study was to identify mutations in the gene encoding for lysosomal beta-glucocerebrosidase (GBA; gene symbol, GBA) in Hungarian patients with Gaucher disease (GD), and to study genotype-phenotype relationships. Genotypes and allele variations in 27 patients with type I GD of 25 unrelated families were studied. Of the 54 mutant alleles, we detected 38 frequent (N370S, 22/54; RecNciI, 8/54; L444P, 8/54) and 9 rare (N188S, R257Q, R285C, G377S, R120W, T323I, 84GG, 1263-1317del and 1263-1317del/RecTL) mutations. In addition, we identified two novel mutations. The N370S/RecNciI genotype found in 8 patients and the N370S/L444P genotype found in 5 patients were the most frequent genotypes in this cohort. In 22 patients the mutations occurred in heterozygosity with the N370S sequence variant, and one patient was homozygous for the L444P mutation. These data suggest that N370S, RecNciI, and L444P are the most prevalent mutations in Hungarian patients with GD. This mutation profile is characteristic for a Caucasian (non-Jewish) population. The c.260G>A and c.999G>A missense mutations are described here for the first time in GD patients contributing to the panel of reported GBA mutations.

We present the third case of Niemann-Pick disease type C without neurological symptoms. The patient was a 53-year-old woman without significant prior health problems who died of acute pulmonary embolism. Autopsy findings of hepatosplenomegaly, lymphadenopathy and ceroid-rich foam cells raised the suspicion of the visceral form of acid sphingomyelinase deficiency (Niemann-Pick disease type B; NPB) or a much rarer disorder, variant adult visceral form of Niemann-Pick disease type C (NPC). To verify the histopathological findings, SMPD1, NPC1 and NPC2 genes were analysed. Two novel sequence variants, c.1997G>A (S666N) and c.2882A>G (N961S) were detected in the NPC1 gene. No pathogenic sequence variants were found either in the SMPD1 gene mutated in NPB or in NPC2 gene. The pathogenicity of both NPC1 variants was supported by their location in regions important for the protein function. Both variations were not found in more than 300 control alleles. Identified sequence variations confirm the diagnosis of the extremely rare adult visceral form of Niemann-Pick disease type C, which is otherwise dominated by neurovisceral symptoms. Although only three patients have been reported, this (most probably underdiagnosed) form of NPC should be considered in differential diagnosis of isolated hepatosplenomegaly with foam cells in adulthood.

X-ALD is a neurological disorder associated with inherited defects in the ABCD1 (ALD) gene located on Xq28 and with impaired peroxisomal very long-chain fatty acid beta-oxidation. We examined the ABCD1 gene in probands from 11 unrelated X-ALD Czech and Slovak families by the direct sequencing of cDNA or genomic PCR products. In 10 families there were 10 different mutations, eight of which were novel. The spectrum of mutations consists of six point mutations, three microdeletions (1bp, 2bp, 4 bp), and one large deletion (229bp). In the 11th family we detected two novel single-base pair substitutions in exon 1 (c.38 A>C and c.649 A>G), both causing amino acid exchanges (N13T and K217E). Expression studies revealed that only K217E is a deleterious mutation, because a plasmid encoding ALDP with K217E was ineffective in the restoration of defective beta-oxidation in X-ALD fibroblasts. The N13T amino acid exchange, on the other hand, did not affect ALDP function. Thus, N13T represents the first polymorphism causing an amino acid exchange in the ABCD1 gene. As this polymorphism was observed neither in 100 control alleles nor in 300 X-ALD patients who have been sequenced so far world-wide, it seems to be very rare or unique. Two additional novel polymorphisms were found by the sequencing of the ABCD1 gene from our patients: c.-59 C/T in the 5'untranslated region and c.2019 C/T (F673F) in exon 10. The frequencies of these two polymorphisms, were 11/150 and 2/150 control alleles, respectively.

UNLABELLED A full-term female offspring of a first and uneventful pregnancy presented at 60 h of life with irritability, tachypnea and respiratory alkalosis progressing to deep coma with clinically dominant circulatory failure, tachycardia and hypotension. Diagnosis of ornithine transcarbamylase (OTC) deficiency was made on the basis of hyperammonaemia, hypocitrullinaemia and extreme hyperexcretion of orotic acid. The baby was treated with peritoneal dialysis, arginine hydrochloride and adequate energy supply. DNA analysis revealed an as of yet unidentified missense mutation in the 6th exon of the OTC gene, resulting in a change of lysine to glutamine at position 210 (K210Q). Her parents were not found to carry this mutation, implying that this mutation may have occurred either de novo in the patient or in a parental germ cell. CONCLUSION An acute neonatal form of OTC deficiency should be considered in the differential diagnosis of coma in female newborns.

Objective To characterize a new lethal fetal and early postnatal variant of adenylosuccinate lyase (ADSL) deficiency. Study design This was a retrospective analysis of 6 patients with very early presentation of ADSL deficiency. Results Most of the 6 patients had impaired intrauterine growth, microcephaly, fetal hypokinesia, and a lack of fetal heart rate variability. Postnatally, they shared severe muscular hypotonia necessitating mechanical ventilation, intractable seizures, and early death. All 6 patients had biochemical evidence of severe (type 1) disease and low residual ADSL activities. All were compound heterozygous for mutations that, based on expression studies, have a pronounced effect on ADSL activity and/or stability. Conclusions ADSL deficiency may present with prenatal growth restriction, fetal and neonatal hypokinesia, and rapidly fatal neonatal encephalopathy. This clinical presentation is associated with genotypes resulting in very low residual enzyme activity.

Background Niemann-Pick disease type C (NPC) is a rare, fatal neurovisceral disorder with autosomal recessive inheritance, and featuring striking clinical variability dependent on the age at onset of neurological symptoms. We report data from a large cohort of 56 Czech patients with NPC diagnosed over a period of 37 years. Methods An observational, retrospective analysis of historic and current clinical and laboratory information was performed among all NPC patients originating from the area of the contemporary Czech Republic and diagnosed between 1975 and 2012. All patients with ≥1 positive diagnostic test and relevant clinical information were included. Data on diagnostic methods (histopathological and/or ultrastructural; biochemical; genetic), clinical status and general information on treatment were collated. Data were examined in accordance with international guidelines for the management of NPC. Results Between 1975 and 1985 diagnoses were based exclusively on specific histopathological findings, often at autopsy. Bone marrow smear (BMS) analyses have proved to be a very specific indicator for NPC and have become an important part of our diagnostic algorithm. Filipin staining and cholesterol esterification assays became the definitive diagnostic tests after 1985 and were applied in 24 of our patients. Since 2005, more and more patients have been assessed using NPC1/NPC2 gene sequencing. Twelve patients were diagnosed with neonatal/early-infantile onset NPC, 13 with the late-infantile onset form, 20 with the juvenile onset form, and nine with the adolescent/adult onset form. Two diagnosed patients remained neurologically asymptomatic at study completion. Nineteen patients were siblings. Causal NPC1 mutations were determined in 38 patients; two identical NPC2 mutations were identified in one patient. In total, 30 different mutations were identified, 14 of which have been confirmed as novel. The frequency of individual mutated NPC1 alleles in our cohort differs compared with previous published data: the most frequent mutant NPC1 allele was p.R1186H (n = 13), followed by p.P1007A (n = 8), p.S954L (n = 8) and p.I1061T (n = 4). Conclusions These data demonstrate the evolution of the diagnostic process in NPC over the last four decades. We estimate the contemporary birth prevalence of NPC in the Czech Republic at 0.93 per 100,000.

The aim of the study was to examine the effect of previous pregnancies and classical cardiovascular risk factors on vascular endothelial function in a group of 264 young and middle-aged women 3 to 11 years postpartum. We examined microvascular functions by peripheral arterial tonometry and EndoPAT 2000 device with respect to a history of gestational hypertension, preeclampsia, fetal growth restriction, the severity of the disease with regard to the degree of clinical signs and delivery date. Besides, we compared Reactive Hyperemia Index (RHI) values and the prevalence of vascular endothelial dysfunction among the groups of women with normal and abnormal values of BMI, waist circumference, systolic and diastolic blood pressures, heart rate, total serum cholesterol levels, serum high-density lipoprotein cholesterol levels, serum low-density lipoprotein cholesterol levels, serum triglycerides levels, serum lipoprotein A levels, serum C-reactive protein levels, serum uric acid levels, and plasma homocysteine levels. Furthermore, we determined the effect of total number of pregnancies and total parity per woman, infertility and blood pressure treatment, presence of trombophilic gene mutations, current smoking of cigarettes, and current hormonal contraceptive use on the vascular endothelial function. We also examined the association between the vascular endothelial function and postpartum whole peripheral blood expression of microRNAs involved in pathogenesis of cardiovascular/cerebrovascular diseases (miR-1-3p, miR-16-5p, miR-17-5p, miR-20a-5p, miR-20b-5p, miR-21-5p, miR-23a-3p, miR-24-3p, miR-26a-5p, miR-29a-3p, miR-92a-3p, miR-100-5p, miR-103a-3p, miR-125b-5p, miR-126-3p, miR-130b-3p, miR-133a-3p, miR-143-3p, miR-145-5p, miR-146a-5p, miR-155-5p, miR-181a-5p, miR-195-5p, miR-199a-5p, miR-210-3p, miR-221-3p, miR-342-3p, miR-499a-5p, and miR-574-3p). A proportion of overweight women (17.94% and 20.59%) and women with central obesity (18.64% and 21.19%) had significantly lower RHI values at 10.0% false positive rate (FPR) both before and after adjustment of the data for the age of patients. At 10.0% FPR, a proportion of women with vascular endothelial dysfunction (RHI &le, 1.67) was identified to have up-regulated expression profile of miR-1-3p (11.76%), miR-23a-3p (17.65%), and miR-499a-5p (18.82%) in whole peripheral blood. RHI values also negatively correlated with expression of miR-1-3p, miR-23a-3p, and miR-499a-5p in whole peripheral blood. Otherwise, no significant impact of other studied factors on vascular endothelial function was found. We suppose that screening of these particular microRNAs associated with vascular endothelial dysfunction may help to stratify a highly risky group of young and middle-aged women that would benefit from early implementation of primary prevention strategies. Nevertheless, it is obvious, that vascular endothelial dysfunction is just one out of multiple cardiovascular risk factors which has only a partial impact on abnormal expression of cardiovascular and cerebrovascular disease associated microRNAs in whole peripheral blood of young and middle-aged women.