Filling the gap: Genetic risk assessment in hypercholesterolemia using LDL-C and LPA genetic scores.

Routine genetic testing in hypercholesterolemia patients reveals a causative monogenic variant in less than 50% of affected individuals. Incomplete genetic characterization is partly due to polygenic factors influencing low-density-lipoprotein-cholesterol (LDL-C). Additionally, functional variants in the LPA gene affect lipoprotein(a)-associated cholesterol concentrations but are difficult to determine due to the complex structure of the LPA gene. In this study we examined whether complementing standard sequencing with the analysis of genetic scores associated with LDL-C and Lp(a) concentrations improves the diagnostic output in hypercholesterolemia patients. 1.020 individuals including 252 clinically diagnosed hypercholesterolemia patients from the FH Register Austria were analyzed by massive-parallel-sequencing of candidate genes combined with array genotyping, identifying nine novel variants in LDLR. For each individual, validated genetic scores associated with elevated LDL-C and Lp(a) were calculated based on imputed genotypes. Integrating these scores especially the score for Lp(a) increased the proportion of individuals with a clearly defined disease etiology to 68.8% compared to 46.6% in standard genetic testing. The study highlights the major role of Lp(a) in disease etiology in clinically diagnosed hypercholesterolemia patients, of which parts are misclassified. Screening for monogenic causes of hypercholesterolemia and genetic scores for LDL-C and Lp(a) permits more precise diagnosis, allowing individualized treatment.

Amp(1q) and tetraploidy are commonly acquired chromosomal abnormalities in relapsed multiple myeloma.

Long-term disease control in multiple myeloma (MM) is typically an unmet medical need, and most patients experience multiple relapses. Fluorescence in situ hybridization (FISH) is the standard technique to detect chromosomal abnormalities (CAs), which are important to estimate the prognosis of MM and the allocation of risk adapted therapies. In advanced stages, the importance of CAs needs further investigation. From 148 MM patients, two or more paired samples, at least one of which was collected at relapse, were analyzed by FISH. Using targeted next-generation sequencing, we molecularly investigated samples harboring relapse-associated CAs. Sixty-one percent of the patients showed a change in the cytogenetic profile during the disease course, including 10% who acquired high-risk cytogenetics. Amp(1q) (≥4 copies of 1q21), driven by an additional increase in copy number in patients who already had 3 copies of 1q21, was the most common acquired CA with 16% affected patients. Tetraploidy, found in 10% of the samples collected at the last time-point, was unstable over the course of the disease and was associated with TP53 lesions. Our results indicate that cytogenetic progression is common in relapsed patients. The relatively high frequency of amp(1q) suggests an active role for this CA in disease progression.

Identifying adult hypophosphatasia in the rheumatology unit.

BACKGROUND: The most frequent manifestation in adult hypophosphatasia (HPP) is musculoskeletal pain. The unspecific nature of its clinical presentation may prevent correct diagnosis. The aim of the study was to assess the prevalence of ALPL mutations in adult patients treated in rheumatological outpatient facilities with evident musculoskeletal symptoms typical for HPP. METHODS: Over a period of 10 years 9,522 patients were screened in the rheumatology outpatient clinic of the Hanusch hospital Vienna. Serum ALP levels ≤ 40 U/L were found in 524 patients. After screening for secondary causes, 73 patients were invited for clinical evaluation. Genetic testing was performed in 23 patients with suspected HPP. Logistic regression models with Firth penalisation were used to estimate the unadjusted and BMI-adjusted association of each clinical factor with HPP. RESULTS: Mutations in the ALPL gene were observed in 57% of genetically screened patients. Arthralgia, fractures, and pain were the leading symptoms in individuals with ALPL mutation. Chondrocalcinosis (OR 29.12; 95% CI 2.02-1593.52) and dental disease (OR 8.33; 95% CI 0.93-143.40) were associated with ALPL mutation, independent of BMI. Onset of symptoms in patients with ALPL mutation was at 35.1 (14.3) years, with a mean duration from symptoms to diagnosis of 14.4 (8.1) years. Bone mineral density (BMD) and trabecular bone score (TBS) as well as bone turnover markers were not indicative for HPP or ALPL mutation. CONCLUSION: HPP can mimic rheumatologic diseases. Thus, HPP should be considered as a possible diagnosis in adult patients presenting with musculoskeletal pain of unknown origin in rheumatology outpatient clinics. In patients with persistently low ALP serum levels and unclear musculoskeletal pain, HPP as the underlying cause has to be considered.

Array genotyping as diagnostic approach in medical genetics.

Genotyping arrays are by far the most widely used genetic tests but are not generally utilized for diagnostic purposes in a medical context. In the present study, we examined the diagnostic value of a standard genotyping array (Illumina Global Screening Array) for a range of indications. Applications included stand-alone testing for specific variants (32 variants in 10 genes), first-tier array variant screening for monogenic conditions (10 different autosomal recessive metabolic diseases), and diagnostic workup for specific conditions caused by variants in multiple genes (suspected familial breast and ovarian cancer, and hypercholesterolemia). Our analyses showed a high analytical sensitivity and specificity of array-based analyses for validated and non-validated variants, and identified pitfalls that require attention. Ethical-legal assessment highlighted the need for a software solution that allows for individual indication-based consent and the reliable exclusion of non-consented results. Cost/time assessment revealed excellent performance of diagnostic array analyses, depending on indication, proband data, and array design. We have implemented some analyses in our diagnostic portfolio, but array optimization is required for the implementation of other indications.

Genotype-Phenotype Comparison in POGZ-Related Neurodevelopmental Disorders by Using Clinical Scoring.

POGZ-related disorders (also known as White-Sutton syndrome) encompass a wide range of neurocognitive abnormalities and other accompanying anomalies. Disease severity varies widely among POGZ patients and studies investigating genotype-phenotype association are scarce. Therefore, our aim was to collect data on previously unreported POGZ patients and perform a large-scale phenotype-genotype comparison from published data. Overall, 117 POGZ patients' genotype and phenotype data were included in the analysis, including 12 novel patients. A severity scoring system was developed for the comparison. Mild and severe phenotypes were compared with the types and location of the variants and the predicted presence or absence of nonsense-mediated RNA decay (NMD). Missense variants were more often associated with mild phenotypes (p = 0.0421) and truncating variants predicted to escape NMD presented with more severe phenotypes (p < 0.0001). Within this group, variants in the prolin-rich region of the POGZ protein were associated with the most severe phenotypes (p = 0.0004). Our study suggests that gain-of-function or dominant negative effect through escaping NMD and the location of the variants in the prolin-rich domain of the protein may play an important role in the severity of manifestations of POGZ-associated neurodevelopmental disorders.

Examining ethical leadership in health care organizations and its impacts on employee work attitudes: an empirical analysis from Austria.

PURPOSE: This study aims to examine the effects of ethical leadership on job satisfaction, affective commitment and burnout of health care employees, considering frustration tolerance and emotional stability as moderating variables. DESIGN/METHODOLOGY/APPROACH: A questionnaire was used to survey health care professionals working in private and public Austrian health-care organizations (hospitals, nursing homes, rehabilitation centers and sanatoriums). The questionnaire consisted of items from well-established scales. The collected data (n = 458) was analyzed using correlation and regression analyzes. FINDINGS: Findings indicated that ethical leadership is significantly positively related to job satisfaction (r = 0.485, p < 0.01) and affective commitment (r = 0.461, p < 0.01) and is significantly negatively related to burnout (r = -0.347, p < 0.01). The results also suggest that frustration tolerance (ß = 0.101, p < 0.1) and emotional stability (ß = 0.093, p < 0.1) moderate the relationship between ethical leadership and burnout. Furthermore, a moderation effect of emotional stability in the ethical leadership and affective commitment relation was indicated. No moderation effect was found for frustration tolerance or emotional stability for the relationship between ethical leadership and job satisfaction. PRACTICAL IMPLICATIONS: Ethical leadership emphasizes the socio-emotional dimension in a leader-employee relationship, which can easily be neglected in times of staff cuts and work overload. Leadership training should include the development of skills in how to visibly act as a moral person, as well as how to set clear ethical standards and communicate them to employees. ORIGINALITY/VALUE: This study adds value to the limited evidence on the beneficial role of ethical leadership in health care settings. In addition, frustration tolerance and emotional stability have not before been investigated as moderators.

An Outbreak of Subclinical Mastitis in a Dairy Herd Caused by a Novel Streptococcus canis Sequence Type (ST55).

The present case report provides data on the phenotypic and genotypic properties of S. canis isolated from nine dairy cows with subclinical mastitis (SCC greater than 200,000 cells/mL in the quarter milk sample, no clinical signs) and from a cat living in the barn and reports the eradication of the pathogen from the herd with an automatic milking system. The isolates were identified using conventional bacteriology, matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF) mass spectrometry (MS) and genetic relationships were investigated by multilocus sequence typing (MLST). Udder health management and hygiene instructions comprised the removal of the carnivores from the barn, strict monitoring of milking hygiene and techniques to avoid new infections via the milking robot, with simultaneous therapy for all infected cows. Phenotypic and genotypic properties of all isolates were identical. MLST revealed a unique sequence type (ST55) and a farmyard cat was identified as the most likely source of the S. canis infection in cows. The simultaneous treatment of all infected cows and management and hygiene improvements lead to a decreased SCC within four weeks.

Tracing Mastitis Pathogens-Epidemiological Investigations of a Pseudomonas aeruginosa Mastitis Outbreak in an Austrian Dairy Herd.

The present study describes an outbreak of Pseudomonas (P.) aeruginosa mastitis in a 20-cow dairy herd where throughout genotyping of isolates reusable udder towels were identified as the source of infection. Sampling of cows during three herd surveys and bacteriological culturing showed that P. aeruginosa was isolated from nine cows with a total of 13 infected quarters. Mastitis occurred as mild clinical or subclinical infection. P. aeruginosa was additionally isolated from a teat disinfectant solution, containing N-(3-aminopropyl)-N-dodécylpropane-1,3-diamine 1 as active component, and microfiber towels used for pre-milking teat preparation. Disc diffusion antimicrobial resistance testing revealed that all isolates were susceptible to piperacillin, piperacillin-tazobactam, ceftazidime, cefepime, aztreonam, imipenem, meropenem, tobramycin, amikacin, and ciprofloxacin. Thirty-two isolates of milk samples and 22 randomly selected isolates of one udder towel and of the teat disinfectant solution were confirmed as P. aeruginosa with matrix-assisted laser desorption, ionization time-of-flight mass spectrometry (MALDI Tof MS). Isolates were further characterized with rep-PCR and randomly amplified polymorphic DNA (RAPD) as well as with multiple locus variable-number tandem repeat analysis (MLVA). Results obtained in this study suggested that one single strain was responsible for the whole outbreak. The transmission occurred throughout a contaminated teat cleaning solution as a source of infection. The farmer was advised to change udder-preparing routine and to cull infected cows.

Clonal dynamics in a composite chronic lymphocytic leukemia and hairy cell leukemia-variant.

Composite lymphoma is the rare simultaneous manifestation of two distinct lymphomas. Chronic lymphocytic leukemia (CLL) has a propensity for occurring in composite lymphomas, a phenomenon that remains to be elucidated. We applied cytogenetics, droplet digital polymerase chain reaction, and massively parallel sequencing to analyze longitudinally a patient with CLL, who 3 years later showed transformation to a hairy cell leukemia-variant (HCL-V). Outgrowth of the IGHV4-34-positive HCL-V clone at the expense of the initially dominant CLL clone with trisomy 12 and MED12 mutation started before CLL-guided treatment and was accompanied by a TP53 mutation, which was already detectable at diagnosis of CLL. Furthermore, deep sequencing of IGH showed a composite lymphoma with presence of both disease components at all analyzed timepoints (down to a minor clone: major clone ratio of ~1:1000). Overall, our analyses showed a disease course that resembled clonal dynamics reported for malignancies with intratumoral heterogeneity and illustrate the utility of deep sequencing of IGH to detect distinct clonal populations at diagnosis, monitor clonal response to therapy, and possibly improve clinical outcomes.

Correction to: Expanding the phenotype of hypomaturation amelogenesis imperfecta due to a novel SLC24A4 variant.

In the pedigree, one of the individuals was marked as unaffected whereas it is heterozygous for the SLC24A4 mutation.

EMQN best practice guidelines for molecular genetic testing and reporting of 21-hydroxylase deficiency.

Molecular genetic testing for congenital adrenal hyperplasia (CAH) due to 21-hydroxylase deficiency (21-OHD) is offered worldwide and is of importance for differential diagnosis, carrier detection and adequate genetic counseling, particularly for family planning. In 2008 the European Molecular Genetics Quality Network (EMQN) for the first time offered a European-wide external quality assessment scheme for CAH (due to 21-OH deficiency). The interest was great and over the last years at about 60 laboratories from Europe, USA and Australia regularly participated in that scheme. These best practice guidelines were drafted on the basis of the extensive knowledge and experience got from those annually organized CAH-schemes. In order to obtain the widest possible consultation with practicing laboratories the draft was therefore circulated twice by EMQN to all laboratories participating in the EQA-scheme for CAH genotyping and was updated by that input. The present guidelines address quality requirements for diagnostic molecular genetic laboratories, as well as criteria for CYP21A2 genotyping (including carrier-testing and prenatal diagnosis). A key aspect of that article is the use of appropriate methodologies (e.g., sequencing methods, MLPA (multiplex ligation dependent probe amplification), mutation specific assays) and respective limitations and analytical accuracy. Moreover, these guidelines focus on classification of variants, and the interpretation and standardization of the reporting of CYP21A2 genotyping results. In addition, the article provides a comprehensive list of common as well as so far unreported CYP21A2-variants.

Expanding the phenotype of hypomaturation amelogenesis imperfecta due to a novel SLC24A4 variant.

OBJECTIVES: Biallelic variants in solute carrier family 24 member 4 (SLC24A4) have been previously reported to cause non-syndromic autosomal recessive amelogenesis imperfecta (AI) of the pigmented hypomaturation type (MIM #615887). We here describe a novel variant in SLC24A4 causing mild enamel hypomaturation defects also in heterozygous individuals. MATERIALS AND METHODS: In the present pedigree analysis, a large consanguineous Syrian family with AI of the hypomaturation type was investigated by clinical and dental evaluation, and exome and Sanger sequencing. Dental histological investigations of seven primary and two permanent teeth were performed. RESULTS: Homozygous variants in SLC24A4 (c.1604G>A; p.Gly535Asp) were identified in five individuals with brown discolorations and irregular pits and grooves of the teeth. Severe attritions, occlusal abfractions, and the radiological lack of contrast between enamel and dentin point out a mineralization defect. Histological dental investigations confirmed the clinical diagnosis of AI of the hypomaturation type. In two heterozygous individuals, a mild hypomaturation defect was present with white and light brown enamel discolorations. CONCLUSIONS: This is the first report of heterozygous SLC24A4 variants causing mild hypomaturation defects, providing confirmatory evidence that the function of SLC24A4 in calcium transport has a crucial role in the maturation stage of amelogenesis. CLINICAL RELEVANCE: The present report is expanding the clinical phenotype of SLC24A4 variants to more severe forms of amelogenesis imperfecta. An autosomal-dominant inheritance pattern with mild clinical phenotypes in heterozygotes has to be considered.

Smith-Lemli-Opitz syndrome - Fetal phenotypes with special reference to the syndrome-specific internal malformation pattern.

BACKGROUND: Autosomal-recessive SLOS is caused by mutations in the DHCR7 gene. It is defined as a highly variable complex of microcephaly with intellectual disability, characteristic facies, hypospadias, and polysyndactyly. Syndrome diagnosis is often missed at prenatal ultrasound and fetal autopsy METHODS: We performed autopsies and DHCR7 gene analyses in eight fetuses suspected of having SLOS and measured cholesterol values in long-term formalin-fixed tissues of an additional museum exhibit RESULTS: Five of the nine fetuses presented classical features of SLOS, including four cases with atrial/atrioventricular septal defects and renal anomalies, and one with additional bilateral renal agenesis and a Dandy-Walker cyst. These cases allowed for diagnosis at autopsy and subsequent SLOS diagnosis in two siblings. Two fetuses were mildly affected and two fetuses showed additional holoprosencephaly. These four cases and the exhibit had escaped diagnosis at autopsy. The case with bilateral renal agenesis presented a novel combination of a null allele and a putative C-terminus missense mutation in the DHCR7 gene CONCLUSIONS: In view of the discrepancy between the prevalence of SLOS among newborns and the carrier frequency of a heterozygous DHCR7 gene mutation, the syndrome-specific internal malformation pattern may be helpful not to miss SLOS diagnosis in fetuses at prenatal ultrasound and fetal autopsy.

Comparison of the population structure of Streptococcus uberis mastitis isolates from Austrian small-scale dairy farms and a Slovakian large-scale farm.

Streptococcus uberis, a major mastitis pathogen associated with intramammary infections (IMI), can be found ubiquitously in the cow's environment. Although Strep. uberis is reported to be susceptible to most antimicrobials, in practice poor responses to treatment and recurrent mastitis are observed. This can be explained by reinfection or by persistence of strains. We hypothesized that among a heterogeneous group of Strep. uberis mastitis isolates, some predominant host-adapted clones might be recurrently isolated from IMI. Therefore, the aim of this pilot study was to determine the Strep. uberis genotype variety found among small-scale dairy herds (127 Austrian dairy farms) and compare this with a large-scale herd (a Slovakian dairy farm). We determined the occurrence and strain diversity of Strep. uberis (n = 309) isolates using molecular analysis. Streptococcus uberis isolates from aseptically collected quarter milk samples were genotypically characterized using pulsed-field gel electrophoresis (PFGE) and multilocus sequence typing. The Strep. uberis strain set covered isolates from 4 Austrian federal areas [Lower Austria (n = 67), Upper Austria (n = 8), Salzburg (n = 51), and Styria (n = 1)] and the Bratislava Region of Slovakia (n = 1). The PFGE analysis resulted in 187 SmaI profiles with 151 unique profiles. Simpson's index of diversity was 0.988. Individual cows (n = 17) harbored up to 3 different PFGE types in the udder. Dairy cows shared distinct PFGE types within a farm. Seven PFGE types were widely distributed among Austrian dairy farms. In the Slovakian farm, 10 predominant PFGE types were recurrently isolated from the same quarters; these genotypes were assigned as persisters. We identified novel sequence types (ST) using multilocus sequence typing related to the global clonal complexes ST5 and ST143. We concluded that Strep. uberis IMI are caused by strains with a wide heterogeneity of PFGE types. This large number of unique subtypes indicates a high diversity of Strep. uberis in the environment. In the large herd, molecular epidemiological results revealed that specific strains might be involved in contagious transmission events and potentially lead to persistence.

Characterization of Staphylococcus Species Isolated from Bovine Quarter Milk Samples.

Staphylococcus (S.) aureus is considered as a major mastitis pathogen, with considerable epidemiological information on such infections while the epidemiology of coagulase-negative staphylococci (CNS) is more controversial. The aim of this study was to use matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) technology for identification of staphylococci isolated from bovine milk at species level and to characterize them in reference to presentation, somatic cell count (SCC), bacterial shedding (cfu) and antimicrobial resistance patterns. A total of 200 staphylococcal isolates (S. aureus n = 100; CNS n = 100) originating from aseptically collected quarter milk samples from different quarters of dairy cows were included in the study. They originated from cases of clinical (CM) and subclinical mastitis (SCM) or were isolated from milk with SCC ≤ 100,000 cells/mL in pure culture. We found staphylococci predominantly in cases of SCM (n = 120). In low-SCC cows, 12 S. aureus and 32 CNS isolates were detected. Eighteen percent of each were associated with CM. Eleven CNS species were identified, S. chromogenes (n = 26) and S. xylosus (n = 40) predominated. CNS, particularly those in low-SCC cows, showed higher MIC90 (minimal inhibitory concentration) values for penicillin, ampicillin, cefoperazone, pirlimycin and marbofloxacin. Based on the present results, a careful interpretation of laboratory results is recommended to avoid antimicrobial therapy of staphylococci without clinical relevance and to ensure prudent use of antimicrobials.

A cohort of 17 patients with kyphoscoliotic Ehlers-Danlos syndrome caused by biallelic mutations in FKBP14: expansion of the clinical and mutational spectrum and description of the natural history.

PurposeIn 2012 we reported in six individuals a clinical condition almost indistinguishable from PLOD1-kyphoscoliotic Ehlers-Danlos syndrome (PLOD1-kEDS), caused by biallelic mutations in FKBP14, and characterized by progressive kyphoscoliosis, myopathy, and hearing loss in addition to connective tissue abnormalities such as joint hypermobility and hyperelastic skin. FKBP14 is an ER-resident protein belonging to the family of FK506-binding peptidyl-prolyl cis-trans isomerases (PPIases); it catalyzes the folding of type III collagen and interacts with type III, type VI, and type X collagens. Only nine affected individuals have been reported to date.MethodsWe report on a cohort of 17 individuals with FKBP14-kEDS and the follow-up of three previously reported patients, and provide an extensive overview of the disorder and its natural history based on clinical, biochemical, and molecular genetics data.ResultsBased on the frequency of the clinical features of 23 patients from the present and previous cohorts, we define major and minor features of FKBP14-kEDS. We show that myopathy is confirmed by histology and muscle imaging only in some patients, and that hearing impairment is predominantly sensorineural and may not be present in all individuals.ConclusionOur data further support the extensive clinical overlap with PLOD1-kEDS and show that vascular complications are rare manifestations of FKBP14-kEDS.

Marathoning with myotonic dystrophy type 2 (proximal myotonic myopathy) and leukopenia.

OBJECTIVES: A mild, slowly progressive course of proximal myotonic myopathy, also known as myotonic dystrophy type 2, over years allowing the patient to continue with extreme sport activity, has been only rarely reported. METHODS: Case report. RESULTS: The patient is a 54-year-old female sport teacher who developed myotonia of the distal upper limbs at the age of 32 years. Over the following 22 years, myotonia spreaded to the entire musculature. Myotonia did not prevent her from doing her job and from marathoning and improved with continuous exercise. Additionally, she had developed hypothyroidism, ovarial cysts, incipient cataract, motor neuropathy, hepatopathy, leukopenia, and mild hyper-CK-emia. A heterozygous CCTG-repeat expansion of 500-9500 was found in the CNBP/ZNF9 gene. At the age of 54 years, she was still performing sport, without presenting with myotonia on clinical examination or having developed other typical manifestations of proximal myotonic myopathy. CONCLUSIONS: This case shows that proximal myotonic myopathy may take a mild course over at least 22 years, that proximal myotonic myopathy with mild myotonia may allow a patient to continue strenuous sport activity, and that continuous physical activity may contribute to the mild course of the disease.

Diagnostic accuracy of a standardized scheme for identification of Streptococcus uberis in quarter milk samples: A comparison between conventional bacteriological examination, modified Rambach agar medium culturing, and 16S rRNA gene sequencing.

Bacteriological examination of milk samples is a prerequisite for pathogen-specific therapy and aids in limiting antimicrobial resistance. The aims of this study were to establish a standardized scheme for reliable Streptococcus uberis identification in routine diagnosis and to evaluate the accuracy of conventional tests and growing patterns of Strep. uberis on a selective medium (modified Rambach agar medium, MRAM) using 16S rRNA gene sequencing analysis as a reference method. We obtained isolates of presumptive Strep. uberis (n = 336) from quarter milk samples of dairy cows with intramammary infections and classified the isolates into 2 clusters using biochemical characterization. In cluster 1 (n = 280), cocci grew as non-hemolytic colonies, hydrolyzing esculin, carrying no Lancefield antigen (A/B/C/D/G) or Christie Atkins Munch-Petersen factor, and their growth was inhibited on an Enterococcus agar. Production of ß-d-galactosidase on MRAM was shown by 257 of the cluster 1 isolates (91.79%), and 16S rRNA gene sequencing verified 271 (96.79%) of the isolates to be Strep. uberis. In 264 isolates (94.29%), MRAM agreed with the sequencing results. In cluster 2 (n = 56), isolates showed different characteristics: 37 (66.07%) were ß-d-galactosidase-positive, and based on 16S sequencing results, 36 (64.29%) were identified correctly as Strep. uberis using biochemical methods. Identification success in this group differed significantly between routine diagnosis and MRAM application: MRAM agreed with sequencing results in 47 isolates (83.93%). To identify Strep. uberis and differentiate it from other lactic acid bacteria in routine diagnosis, we suggest using catalase reaction, hemolysis, esculin hydrolysis, and growth on enterococci agar. Isolates that show a typical biochemical profile can be identified satisfactorily with these tests. For Strep. uberis isolates with divergent patterns, application of MRAM as a follow-up test increased the diagnostic accuracy to 94.64%.