Recent advances in genome sequencing technologies provide unprecedented opportunities to characterize individual genomic landscapes and identify mutations relevant for diagnosis and therapy. ...Specifically, whole-exome sequencing using next-generation sequencing (NGS) technologies is gaining popularity in the human genetics community due to the moderate costs, manageable data amounts and straightforward interpretation of analysis results. While whole-exome and, in the near future, whole-genome sequencing are becoming commodities, data analysis still poses significant challenges and led to the development of a plethora of tools supporting specific parts of the analysis workflow or providing a complete solution. Here, we surveyed 205 tools for whole-genome/whole-exome sequencing data analysis supporting five distinct analytical steps: quality assessment, alignment, variant identification, variant annotation and visualization. We report an overview of the functionality, features and specific requirements of the individual tools. We then selected 32 programs for variant identification, variant annotation and visualization, which were subjected to hands-on evaluation using four data sets: one set of exome data from two patients with a rare disease for testing identification of germline mutations, two cancer data sets for testing variant callers for somatic mutations, copy number variations and structural variations, and one semi-synthetic data set for testing identification of copy number variations. Our comprehensive survey and evaluation of NGS tools provides a valuable guideline for human geneticists working on Mendelian disorders, complex diseases and cancers.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Abstract
STUDY QUESTION
When should cystic fibrosis transmembrane conductance regulator (CFTR) mutation analysis be recommended in infertile men based on andrological findings?
SUMMARY ANSWER
CFTR ...mutation analysis is recommended in all men with unexplained azoospermia in the presence of normal gonadotropin levels.
WHAT IS KNOWN ALREADY
While 80–97% of men with congenital bilateral absence of the vas deferens (CBAVD) are thought to carry CFTR mutations, there is uncertainty about the spectrum of clinical and andrological abnormalities in infertile men with bilallelic CFTR mutations. This information is relevant for evidence-based recommendations to couples requesting assisted reproduction.
STUDY DESIGN, SIZE, DURATION
We studied the andrological findings of patients with two CFTR mutations who were examined in one of the cooperating fertility centres in Germany and Austria. In the period of January till July 2019, the completed and anonymized data sheets of 78 adult male patients were returned to and analysed by the project leader at the Institute of Human Genetics in Innsbruck, Austria.
PARTICIPANTS/MATERIALS, SETTING, METHODS
Minimum study entry criteria were the presence of two (biallelic) CFTR mutations and results of at least one semen analysis. Andrological assessments were undertaken by standardized data sheets and compared with normal reference values. Seventy-one patients were eligible for the study (n = 30, 42% from Germany, n = 26, 37% from Austria, n = 15, 21% other nations).
MAIN RESULTS AND THE ROLE OF CHANCE
Gonadotropin levels (FSH, LH) were normal, 22% of patients had reduced testosterone values. Mean right testis volume was 23.38 ml (SD 8.77), mean left testis volume was 22.59 ml (SD 8.68) and thereby statistically increased compared to normal (P < 0.01). although the means remained in the reference range of 12–25 ml. Semen analysis revealed azoospermia in 70 of 71 (99%) patients and severe oligozoospermia <0.1 × 106/ml in one patient. Four semen parameters, i.e. ejaculate volume, pH, α-glucosidase and fructose values, were significantly reduced (P < 0.01). Only 18% of patients had a palpatory and sonographically diagnosed CBAVD, while in 31% the diagnosis of CBAVD was uncertain, in 12% patients, the vas deferens was present but hypoplastic, and in 39% the vas deferens was normally present bilaterally. Seminal vesicles were not detectable in 37% and only unilaterally present in 37% of patients. Apart from total testes volume, clinical findings were similar in patients with two confirmed pathogenic CFTR mutations (Group I) compared with patients who carried one pathogenic mutation and one CFTR variant of unknown significance (Group II).
LIMITATIONS, REASONS FOR CAUTION
We could not formally confirm the in trans position of genetic variants in most patients as no family members were available for segregation studies. Nonetheless, considering that most mutations in our study have been previously described without other rare variants in cis, and in view of the compatible andrological phenotype, it is reasonable to assume that the biallelic genotypes are correct.
WIDER IMPLICATIONS OF THE FINDINGS
Our study reveals that CFTR mutation analysis has a broader indication than just the absence of the vas deferens. We recommend to completely sequence the CFTR gene if there is a suspicion of obstructive azoospermia, and to extend this analysis to all patients with unexplained azoospermia in the presence of normal gonadotropin levels.
STUDY FUNDING/COMPETING INTEREST(S)
German Research Foundation Clinical Research Unit ‘Male Germ Cells: from Genes to Function’ (DFG CRU326, grants to F.T.). There are no conflicts of interest to declare.
TRIAL REGISTRATION NUMBER
N/A.
Periodontal Ehlers-Danlos Syndrome (pEDS) is a rare autosomal dominant type of EDS characterised by severe early-onset periodontitis, lack of attached gingiva, pretibial plaques, joint hypermobility ...and skin hyperextensibility as per the 2017 International EDS Classification. In 2016, deleterious pathogenic heterozygous variants were identified in
and
, which encode components of the complement system.
Individuals with a clinical suspicion of pEDS were clinically and molecularly assessed through the National EDS Service in London and Sheffield and in genetic services in Austria, Sweden and Australia. Transmission electron microscopy and fibroblast studies were performed in a small subset of patients.
A total of 21 adults from 12 families were clinically and molecularly diagnosed with pEDS, with
variants in all families. The age at molecular diagnosis ranged from 21-73 years (mean 45 years), male: female ratio 5:16. Features of easy bruising (90%), pretibial plaques (81%), skin fragility (71%), joint hypermobility (24%) and vocal changes (38%) were identified as well as leukodystrophy in 89% of those imaged.
This cohort highlights the clinical features of pEDS in adults and contributes several important additional clinical features as well as novel deleterious variants to current knowledge. Hypothetical pathogenic mechanisms which may help to progress understanding and management of pEDS are also discussed.
Published data on treatment of fatty acid oxidation defects are scarce. Treatment recommendations have been developed on the basis of observations in 75 patients with long-chain fatty acid oxidation ...defects from 18 metabolic centres in Central Europe. Recommendations are based on expert practice and are suggested to be the basis for further multicentre prospective studies and the development of approved treatment guidelines. Considering that disease complications and prognosis differ between different disorders of long-chain fatty acid oxidation and also depend on the severity of the underlying enzyme deficiency, treatment recommendations have to be disease-specific and depend on individual disease severity. Disorders of the mitochondrial trifunctional protein are associated with the most severe clinical picture and require a strict fat-reduced and fat-modified (medium-chain triglyceride-supplemented) diet. Many patients still suffer acute life-threatening events or long-term neuropathic symptoms despite adequate treatment, and newborn screening has not significantly changed the prognosis for these severe phenotypes. Very long-chain acyl-CoA dehydrogenase deficiency recognized in neonatal screening, in contrast, frequently has a less severe disease course and dietary restrictions in many patients may be loosened. On the basis of the collected data, recommendations are given with regard to the fat and carbohydrate content of the diet, the maximal length of fasting periods and the use of l-carnitine in long-chain fatty acid oxidation defects.
Psychotropic medications target glycogen synthase kinase 3β (GSK3β), but the functional integration with other factors relevant for drug efficacy is poorly understood. We discovered that the ...suggested psychiatric risk factor FK506 binding protein 51 (FKBP51) increases phosphorylation of GSK3β at serine 9 (pGSK3β(S9)). FKBP51 associates with GSK3β mainly through its FK1 domain; furthermore, it also changes GSK3β's heterocomplex assembly by associating with the phosphatase PP2A and the kinase cyclin-dependent kinase 5. FKBP51 acts through GSK3β on the downstream targets Tau, β-catenin and T-cell factor/lymphoid enhancing factor (TCF/LEF). Lithium and the antidepressant (AD) paroxetine (PAR) functionally synergize with FKBP51, as revealed by reporter gene and protein association analyses. Deletion of FKBP51 blunted the PAR- or lithium-induced increase in pGSK3β(S9) in cells and mice and attenuated the behavioral effects of lithium treatment. Clinical improvement in depressive patients was predicted by baseline GSK3β pathway activity and by pGSK3β(S9) reactivity to ex vivo treatment of peripheral blood mononuclear lymphocytes with lithium or PAR. In sum, FKBP51-directed GSK3β activity contributes to the action of psychotropic medications. Components of the FKBP51-GSK3β pathway may be useful as biomarkers predicting AD response and as targets for the development of novel ADs.
We have investigated the correlation between genotype and phenotype in a large number of patients with glutaric aciduria type I (GA I). The deficiency of glutaryl‐CoA dehydrogenase has been confirmed ...in the Rigshospitalet's laboratory in 215 patients since 1975. Most of the patients were of European ancestry. Complete absence of enzyme activity was found in more than half of the patients, while 34% of patients had a residual activity up to 5% and a few patients had a residual activity of 5–15%. In four exceptional cases, a very high residual activity of up to 30% was found. Enzyme studies are thus a reliable method for confirming the diagnosis of GA I, although it may be difficult to distinguish exceptional ‘mild’ cases from heterozygous carriers for GA I. Three of the patients with very high residual activity are compound heterozygous for the missense mutations R227P and V400M, both of which are associated with residual enzyme activity of 8–10% in homozygous patients. Patients with a mild mutation on at least one chromosome frequently show unusual biochemical findings such as low or normal urinary excretion of glutaric acid and mild or only slightly increased excretion of 3‐hydroxyglutaric acid. In contrast, patients with severe mutations such as R402W or A293T on both alleles have no residual activity and show the typical urinary metabolite pattern. Clinical data were available for a subgroup of 79 patients. No correlation with the biochemical phenotype or the genotype could be established.
At present, long-chain fatty acid oxidation (FAO) defects are diagnosed in a number of countries by newborn screening using tandem mass spectrometry. In the majority of cases, affected newborns are ...asymptomatic at time of diagnosis and acute clinical presentations can be avoided by early preventive measures. Because evidence-based studies on management of long-chain FAO defects are lacking, we carried out a retrospective analysis of 75 patients from 18 metabolic centres in Germany, Switzerland, Austria and the Netherlands with special regard to treatment and disease outcome. Dietary treatment is effective in many patients and can prevent acute metabolic derangements and prevent or reverse severe long-term complications such as cardiomyopathy. However, 38% of patients with very long-chain acyl-CoA dehydrogenase (VLCAD) deficiency had intermittent muscle weakness and pain despite adhering to therapy. Seventy-six per cent of patients with disorders of the mitochondrial trifunctional protein (TFP)-complex including long-chain 3-hydroxyacyl-CoA dehydrogenase (LCHAD) deficiency, had long-term myopathic symptoms. Of these, 21% had irreversible peripheral neuropathy and 43% had retinopathy. The main principle of treatment was a fat-reduced and fat-modified diet. Fat restriction differed among patients with different enzyme defects and was strictest in disorders of the TFP-complex. Patients with a medium-chain fat-based diet received supplementation of essential long-chain fatty acids. l-Carnitine was supplemented in about half of the patients, but in none of the patients with VLCAD deficiency identified by newborn screening. In summary, in this cohort the treatment regimen was adapted to the severity of the underlying enzyme defect and thus differed among the group of long-chain FAO defects.
In humans, we reported an association of a certain allele of carnosinase gene with reduced carnosinase activity and absence of nephropathy in diabetic patients. CN1 degrades histidine dipeptides such ...as carnosine and anserine. Further, we and others showed that treatment with carnosine improves renal function and wound healing in diabetic mice and rats. We now investigated the effects of carnosine treatment alone and in combination with ACE inhibition, a clinically established nephroprotective drug in diabetic nephropathy.
Male Sprague–Dawley rats were injected i.v. with streptozotocin (STZ) to induce diabetes. After 4weeks, rats were unilaterally nephrectomized and randomized for 24weeks of treatment with carnosine, lisinopril or both.
Renal CN1 protein concentrations were increased under diabetic conditions which correlated with decreased anserine levels. Carnosine treatment normalized CN1 abundance and reduced glucosuria, blood concentrations of glycosylated hemoglobin (HbA1c), carboxyl-methyl lysine (CML), N-acetylglucosamine (GlcNac; all p<0.05 vs. non-treated STZ rats), reduced cataract formation (p<0.05) and urinary albumin excretion (p<0.05), preserved podocyte number (p<0.05) and normalized the increased renal tissue CN1 protein concentration. Treatment with lisinopril had no effect on HbA1C, glucosuria, cataract formation and CN1 concentration, but reduced albumin excretion rate more effectively than carnosine treatment (p<0.05). Treatment with both carnosine and lisinopril combined the effects of single treatment, albeit without additive effect on podocyte number or albuminuria.
Increased CN1 amount resulted in decreased anserine levels in the kidney. Both carnosine and lisinopril exert distinct beneficial effects in a standard model of diabetic nephropathy. Both drugs administered together combine the respective effects of single treatment, albeit without exerting additive nephroprotection.
•Carnosine treatment has renoprotective function in diabetic rats.•Renoprotection differs from the effect of the ACE inhibitor lisinopril.•Carnosinase activity correlates with anserine levels in the kidney.•Carnosine and lisinopril together combine the effect of single treatment.
Duchenne muscular dystrophy (DMD) and Becker muscular dystrophy (BMD) are common X-chromosomal recessive disorders caused by mutations in the dystrophin gene. Using the novel multiplex ...ligation-dependent probe amplification (MLPA) method we performed retrospective and prospective analyses in a total of 193 individuals. Deletions or duplications were identified in 14 out of 90 families previously tested negative by multiplex PCR or FISH analysis. Partially incorrect results were subsequently identified in two families: the loss of exon 38 signal in one case was due to a p.Q1802X nonsense mutation, whilst in another patient an apparent deletion of exon 37 (coinciding with a duplication of exons 46-53) was caused by a p.R1735C polymorphism. In one case we found a complex rearrangement involving a duplication of two regions: dupEX45-48 and dupEX54-55. We conclude that MLPA is a highly sensitive and rapid alternative to multiplex PCR. It can be used on blood samples, chorionic villi and paraffin-embedded tissue. The ease of detection of duplications and the application for female carrier analysis are clearly the main advantages of the method. However, apparent single exon deletions detected by MLPA should be checked by an independent method. Complex rearrangements such as double mutations on the same allele are rare.
Summary
Autosomal recessive congenital ichthyosis (ARCI) caused by mutations in CYP4F22 is very rare. CyP4F22, a protein of the cytochrome‐P450 family 4, encodes an epidermal ω‐hydroxylase decisive ...in the formation of acylceramides, which is hypothesized to be crucial for skin‐barrier function. We report a girl with consanguineous parents presenting as collodion baby with contractures of the great joints and palmoplantar hyperlinearity. In the course of the disease she developed fine scaling of the skin with erythroderma, the latter disappearing until the age of 6 months. Her sister showed a generalized fine‐scaling phenotype, and, interestingly, was born without a collodion membrane. The analysis of all known candidate genes for ARCI in parallel with a next‐generation sequencing approach using a newly designed dermatogenetics gene panel revealed a previously unknown homozygous splice‐site mutation c.549+5G>C in CYP4F22 in both girls, confirming the diagnosis of ARCI. Ultrastructural analysis by transmission electron microscopy in both patients showed epidermal hyperplasia, orthohyperkeratosis with persistence of corneodesmosomes into the outer stratum corneum layers, fragmented and disorganized lamellar lipid bilayers, which could be ascribed to inhomogeneous lamellar body secretion, as well as lamellar body and lipid entombment in the corneocytes. These findings correlated with increased transepidermal water loss on the functional level. For the first time, we report a collodion baby phenotype and epidermal barrier impairment in CyP4F22‐deficient epidermis at both the ultrastructural and functional level, and corroborate the importance of CyP4F22 for epidermal maturation and barrier function.
What's already known about this topic?
Autosomal recessive congenital ichthyosis (ARCI) caused by CYP4F22 mutations is very rare.
There is evidence that CyP4F22 plays a role in the 12(R)‐lipoxygenase pathway and therefore is important for skin‐barrier function.
What does this study add?
We report a novel homozygous splice‐site mutation c.549+5G>C in CYP4F22 in two sisters with ARCI, presenting with and without a collodion membrane at birth.
Transmission electron microscopy reveals epidermal barrier abnormalities in ARCI with CYP4F22 mutations.
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Linked Comment: Hausser. Br J Dermatol 2017; 176:863–864
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