[Rational use of genetic tests in internal medicine : Possibilities and limitations of next generation sequencing diagnostics]. / Rationaler Einsatz genetischer Tests in der Inneren Medizin : Möglichkeiten und Limitationen der Next-generation-sequencing-Diagnostik.

BACKGROUND: New methods of molecular genetic diagnostics enable a more comprehensive genetic analysis of patients. OBJECTIVES: Rational use and benefits of molecular genetic testing in patients with various internal diseases. METHOD: Evaluation of topic-related literature, discussion of own experiences, as well as consideration of current guidelines. RESULTS: New genetic tests, such as next generation sequencing (NGS), improve the diagnosis of hereditary diseases; however, the use of this technology also leads to additional findings, which must be carefully considered. CONCLUSION: The rational use of genetic tests is a benefit for patients and can significantly influence the prevention and treatment of a disease. The increasing complexity of genetic findings requires interdisciplinary approaches involving human genetics, internal medicine, and other disciplines.

Molecular and clinical studies in 8 patients with Temple syndrome.

Temple syndrome (TS14, #616222) is a rare imprinting disorder characterised by phenotypic features including pre- and postnatal growth retardation, muscular hypotonia and feeding difficulties in infancy, early puberty and short stature with small hands and feet and often truncal obesity. It is caused by maternal uniparental disomies, paternal deletions and primary imprinting defects that affect the chromosomal region 14q32 and lead to a disturbed expression of imprinted genes in this region. Here, we present detailed clinical data of 8 patients with Temple syndrome, 4 with an imprinting defect, 2 with an imprinting defect in a mosaic state as well as 1 complete and 1 segmental maternal uniparental disomy of chromosome 14.

Recent Advances in Imprinting Disorders.

Imprinting disorders (ImpDis) are a group of currently 12 congenital diseases with common underlying (epi)genetic etiologies and overlapping clinical features affecting growth, development and metabolism. In the last years it has emerged that ImpDis are characterized by the same types of mutations and epimutations, i.e. uniparental disomies, copy number variations, epimutations, and point mutations. Each ImpDis is associated with a specific imprinted locus, but the same imprinted region can be involved in different ImpDis. Additionally, even the same aberrant methylation patterns are observed in different phenotypes. As some ImpDis share clinical features, clinical diagnosis is difficult in some cases. The advances in molecular and clinical diagnosis of ImpDis help to circumvent these issues, and they are accompanied by an increasing understanding of the pathomechanism behind them. As these mechanisms have important roles for the etiology of other common conditions, the results in ImpDis research have a wider effect beyond the borders of ImpDis. For patients and their families, the growing knowledge contributes to a more directed genetic counseling of the families and personalized therapeutic approaches.

Maternal uniparental disomy of chromosome 16 [upd(16)mat]: clinical features are rather caused by (hidden) trisomy 16 mosaicism than by upd(16)mat itself.

Maternal uniparental disomy of chromosome 16 [upd(16)mat] as the result of trisomy 16 is one of the most frequently reported uniparental disomies in humans, but a consistent phenotype is not obvious. Particularly, it is difficult to discriminate between features resulting from upd(16)mat and mosaic trisomy 16. By evaluating literature data (n = 74) and three own cases we aimed to determine whether the clinical features are due to upd(16)mat or to trisomy 16 mosaicism. While in single cases the clinical symptoms were caused by homozygosity of autosomal recessive mutations on chromosome 16, it turned out that clinical features in upd(16)mat are caused by (hidden) trisomy 16 mosaicism and a specific chromosome 16-associated imprinting disorder does not exist. In trisomy 16/upd(16)mat pregnancies, the management should be based on the ultrasound results and on the clinical course of the pregnancy. In fact, mosaic trisomy 16 pregnancies require a close monitoring because of the higher risk for hypertensive disorders. Postnatal testing for upd(16)mat should be considered in case of homozygosity for an autosomal-recessive mutation, in individuals carrying chromosome 16 aberrations and in phenotypes comprising features of the trisomy 16/upd(16)mat spectrum. Finally, upd(16)mat probably represents a bioindicator for a hidden trisomy 16 mosaicism.

NSD1 duplication in Silver-Russell syndrome (SRS): molecular karyotyping in patients with SRS features.

Silver-Russell syndrome (SRS) is a growth retardation syndrome characterized by intrauterine and postnatal growth retardation, relative macrocephaly and protruding forehead, body asymmetry and feeding difficulties. Nearly 50% of cases show a hypomethylation in 11p15.5, in 10% maternal uniparental disomy of chromosome 7 is present. A significant number of patients with SRS features also exhibit chromosomal aberrations. We analyzed 43 individuals referred for SRS genetic testing by molecular karyotyping. Pathogenic variants could be detected in five of them, including a NSD1 duplication in 5q35 and a 14q32 microdeletion. NSD1 deletions are detectable in overgrowth disorders (Sotos syndrome and Beckwith-Wiedemann syndrome), whereas NSD1 duplications are associated with growth retardation. The 14q32 deletion is typically associated with Temple syndrome (TS14), but the identification of a patient in our cohort reflects the clinical overlap between TS14 and SRS. As determination of molecular subtypes is the basis for a directed counseling and therapy, the identification of pathogenic variants in >10% of the total cohort of patients referred for SRS testing and in >16% of characteristic individuals with the characteristic SRS phenotype confirms the need to apply molecular karyotyping in this cohort.

Syndromic ciliopathies: From single gene to multi gene analysis by SNP arrays and next generation sequencing.

Joubert syndrome (JS) and related disorders (JSRD), Meckel syndrome (MKS) and Bardet-Biedl syndrome (BBS) are autosomal recessive ciliopathies with a broad clinical and genetic overlap. In our multiethnic cohort of 88 MKS, 61 JS/JSRD and 66 BBS families we performed genetic analyses and were able to determine mutation frequencies and detection rates for the most frequently mutated MKS genes. On the basis of determined mutation frequencies, a next generation gene panel for JS/JSRD and MKS was established. Furthermore 35 patients from 26 unrelated consanguineous families were investigated by SNP array-based homozygosity mapping and subsequent DNA sequencing of known candidate genes according to runs of homozygosity size in descending order. This led to the identification of the causative homozygous mutation in 62% of unrelated index cases. Based on our data we discuss various strategies for diagnostic mutation detection in the syndromic ciliopathies JS/JSRD, MKS and BBS.

Alcohol Consumption in Healthy OPRM1 G Allele Carriers and Its Association with Impulsive Behavior.

AIMS: A link between alcohol use disorders (AUD) and impulsivity is well established. As there is evidence for the heritability of AUD, the investigation of the underlying genetic disposition for both conditions is an important issue. An association between AUD and a coding single nucleotide polymorphism (SNP) (rs1799971 encoding an Asn40Asp amino acid substitution, A118G) within the µ-opioid receptor 1 gene (OPRM1) has been reported. Therefore we tested the association between the OPRM1 A118G polymorphism and drinking as well as impulsive behavior in social drinkers. METHODS: A total of 214 healthy male social drinkers were recruited. Each participant was genotyped for the OPRM1 A118G variant. Alcohol use was assessed with items of the Alcohol Use Disorders Identification Test (AUDIT). Impulsivity was assessed using the UPPS impulsive behavior scale. For statistical analyses, we considered correlations, t-tests and ordinal regression models using SPSS V21. RESULTS: In total, 49 out of 214 participants were carriers of the OPRM1 118G allele. On average the OPRM1 118G carriers showed a slightly higher propensity for alcohol drinking. Higher drinking frequency among the G allele carriers was linked with higher urgency and perseveration subscores of impulsivity. CONCLUSION: Our results suggest a genetically influenced higher propensity for alcohol drinking among social drinkers carrying the 118G allele of the OPRM1 gene. The positive correlation between urgency and a higher drinking frequency among the OPRM1 118G hint towards a functional meaning of the opioid system in the regulation of impulsivity.

Twenty-one years to the right diagnosis - clinical overlap of Simpson-Golabi-Behmel and Beckwith-Wiedemann syndrome.

Clinical overlap makes the diagnosis of overgrowth syndromes challenging. Clinical overlap exists between Simpson-Golabi-Behmel syndrome (SGBS) and Beckwith-Wiedemann syndrome (BWS) which share pre- and postnatal overgrowth, macroglossia, umbilical hernia, organomegaly, ear lobe creases, and occurrence of embryonal tumors as characteristic features. Based on the clinical history of a patient, who was diagnosed with BWS shortly after birth and reassessed and rediagnosed with SGBS at age 21 years, particular attention should be paid to developing facial dysmorphia. In addition, we delineate further clinical findings that may allow differentiation between both conditions.

[Genetics and epigenetics. Explanatory approaches for (gender-specific) mechanisms of disease development]. / Genetik und Epigenetik. Erklärungsansätze für (geschlechtsspezifische) Mechanismen der Krankheitsentstehung.

Whereas the central role of DNA as the carrier of genetic information has long been well known, the impact of epigenetic mechanisms as mediators between genes and environment is now becoming increasingly clear. Epigenetics helps explain the partially reversible interplay between gene function and environment and even permits observation of the transgenerational transmission of epigenetic modifications. Of special interest are gender-specific mechanisms of gene regulation which, among others, offer an explanation for gender differences in human diseases. Since the study of epigenetic mechanisms and their impact on the etiology of common diseases is in its infancy, it is too early to draw general conclusions from the current state of knowledge. Moreover, completely new strategies are needed to research these effects. In addition to molecular findings, definitions of specific phenotypes are required, including biographic data of affected individuals and their ancestors. Epigenetics needs to be viewed in the context of the theory of evolution, classical genetics, and environmental research. Its aim is not to substitute the knowledge in these disciplines, but rather to provide a key to link their findings, thereby opening up new possibilities in terms of interpretation and understanding of gender differences in medicine. If these epigenetic mechanisms are better understood, particularly in terms of specific diseases, it is conceivable that these disorders could be influenced and treated in a more targeted manner in the future.

Adult height and epigenotype in children with Silver-Russell syndrome treated with GH.

AIMS: To compare adult heights of GH-treated and GH-untreated patients with Silver-Russell syndrome (SRS) who were epigenotyped. METHODS: This was a nonrandomized retrospective study with matched controls at a single center. Molecular analysis of 32 out of 37 GH-treated patients (16 females) revealed IGF2-H19 epimutations in 12 and maternal uniparental disomy of chromosome 7 (matUPD7) in 5 patients; 15 were negative. At start of GH, mean age was 7.2 years and mean height -3.34 standard deviation score (SDS). Mean GH dose used was 51 µg/kg·day, mean duration of therapy was 5.6 years. Puberty was blocked by GnRH analogs in 16 patients. The untreated group comprised 13 individuals (5 females, mean age 6.8 years and mean height -3.34 SDS). End points were adult height and overall height gain. RESULTS: GH-treated patients reached an adult height of -2.12 ± 0.98 SDS gaining 1.22 SDS in comparison to baseline. Adult height SDS of the untreated was -3.13 ± 1.37 SDS. The matched treated patients were significantly taller than their untreated counterparts. Outcome was dependent on height at start of GH and duration of therapy. Height gain was highest in the shortest patients. CONCLUSIONS: GH improved adult height in SRS to a comparable degree as reported in nonsyndromic SGA children. A trend toward a better outcome in matUPD7 needs confirmation in larger cohorts.

Haploinsufficiency of ANKRD11 (16q24.3) Is Not Obligatorily Associated with Cognitive Impairment but Shows a Clinical Overlap with Silver-Russell Syndrome.

Microdeletions in 16q24.3 are associated with intellectual disability and a specific phenotype, e.g. short stature and a prominent forehead. The 16q24.3 microdeletion syndrome shows a broad phenotypic overlap with the KBG syndrome, which is caused by mutations within the ANKRD11 gene. Furthermore, both KBG and the 16q24.3 microdeletion syndromes show clinical findings reminiscent of Silver-Russell syndrome (SRS), an imprinting disorder characterized by severe primordial growth retardation. In a cohort of patients referred as SRS, we previously identified a 16q24.3 deletion, but at that time, only patients with larger imbalances in 16q24.3 and intellectual disability had been published. Considering the recent description of the ANKRD11 gene as the causative factor for the 2 16q24.3-associated disorders, we now classified our patient as a 16q24.3 microdeletion syndrome patient exhibiting some characteristic features but normal intelligence. Our case illustrates the broad clinical spectrum associated with microdeletions, and we confirm that the 16q24.3 microdeletion syndrome is a further microdeletion syndrome with very variable expressivity. Indeed, our case is the first 16q24.3 patient of normal intelligence, but we assume that this variant is present in further mentally healthy probands which have not yet been tested. In conclusion, the detection of the 16q24.3 deletion in a proband of unremarkable intellectual capacities once again illustrates the need to perform molecular karyotyping in dysmorphic patients with normal intelligence.

Silver-Russell patients showing a broad range of ICR1 and ICR2 hypomethylation in different tissues.

In all known congenital imprinting disorders an association with aberrant methylation or mutations at specific loci was well established. However, several patients with transient neonatal diabetes mellitus (TNDM), Silver-Russell syndrome (SRS) and Beckwith-Wiedemann syndrome (BWS) exhibiting multilocus hypomethylation (MLH) have meanwhile been described. Whereas TNDM patients with MLH show clinical symptoms different from carriers with isolated 6q24 aberrations, MLH carriers diagnosed as BWS or SRS present only the syndrome-specific features. Interestingly, SRS and BWS patients with nearly identical MLH patterns in leukocytes have been identified. We now report on the molecular findings in DNA in three SRS patients with hypomethylation of both 11p15 imprinted control regions (ICRs) in leukocytes. One patient was a monozygotic (MZ) twin, another was a triplet. While the hypomethylation affected both oppositely imprinted 11p15 ICRs in leukocytes, in buccal swab DNA only the ICR1 hypomethylation was visible in two of our patients. In the non-affected MZ twin of one of these patients, aberrant methylation was also present in leukocytes but neither in buccal swab DNA nor in skin fibroblasts. Despite mutation screening of several factors involved in establishment and maintenance of methylation marks including ZFP57, MBD3, DNMT1 and DNMT3L the molecular clue for the ICR1/ICR2 hypomethylation in our patients remained unclear. Furthermore, the reason for the development of the specific SRS phenotype is not obvious. In conclusion, our data reflect the broad range of epimutations in SRS and illustrate that an extensive molecular and clinical characterization of patients is necessary.

Effects of a CACNA1C genotype on attention networks in healthy individuals.

BACKGROUND: Recent genetic studies found the A allele of the variant rs1006737 in the alpha 1C subunit of the L-type voltage-gated calcium channel (CACNA1C) gene to be over-represented in patients with psychosis, including schizophrenia, bipolar disorder and major depressive disorder. In these disorders, attention deficits are among the main cognitive symptoms and have been related to altered neural activity in cerebral attention networks. The particular effect of CACNA1C on neural function, such as attention networks, remains to be elucidated. METHOD: The current event-related functional magnetic resonance imaging (fMRI) study investigated the effect of the CACNA1C gene on brain activity in 80 subjects while performing a scanner-adapted version of the Attention Network Test (ANT). Three domains of attention were probed simultaneously: alerting, orienting and executive control of attention. RESULTS: Risk allele carriers showed impaired performance in alerting and orienting in addition to reduced neural activity in the right inferior parietal lobule [Brodmann area (BA) 40] during orienting and in the medial frontal gyrus (BA 8) during executive control of attention. These areas belong to networks that have been related to impaired orienting and executive control mechanisms in neuropsychiatric disorders. CONCLUSIONS: Our results suggest that CACNA1C plays a role in the development of specific attention deficits in psychiatric disorders by modulation of neural attention networks.

Medial temporal lobe dysfunction during encoding and retrieval of episodic memory in non-demented APOE epsilon4 carriers.

Presence of the apolipoprotein E (APOE) epsilon4 allele is linked to an increased risk to develop Alzheimer's dementia (AD). However, there are controversial data concerning the impact of the APOE genotype on cognitive functioning and brain activity in healthy subjects. We used event-related functional magnetic resonance imaging (fMRI) to investigate the effects of APOE genotype on spatial contextual memory encoding and retrieval success in healthy older adults. Eighteen subjects (eight APOE4 heterozygotes (epsilon4+) and 10 non-carriers (epsilon4-), mean age 60.0+/-5.0 years) were included in the present analysis. Behaviorally, epsilon4+ subjects performed significantly worse than epsilon4- subjects in item memory and spatial context retrieval. fMRI data revealed that epsilon4+ subjects, compared to epsilon4-subjects, predominantly showed an increase of neural activity specific to encoding of items and their spatial context in prefrontal, temporal and parietal regions. In contrast, epsilon4+ subjects showed activity decreases in the right amygdala during successful item recognition and in the prefrontal cortex bilaterally during spatial context retrieval when compared to epsilon4- subjects. While the activity increases during encoding may reflect compensatory activity in the attempt to maintain normal performance, the decreases during retrieval indicate incipient neural decline in epsilon4+ subjects. These data highlight that preclinical ApoE-related changes in neural activity are not unidirectional but dissociate depending on the memory phase, i.e., encoding or retrieval.

Submicroscopic chromosomal imbalances in idiopathic Silver-Russell syndrome (SRS): the SRS phenotype overlaps with the 12q14 microdeletion syndrome.

Silver-Russell syndrome (SRS) is a heterogeneous disorder associated with intrauterine and postnatal growth restriction, body asymmetry, a relative macrocephaly, a characteristic triangular face and further dysmorphisms. In about 50% of patients, genetic/epigenetic alterations can be detected: >38% of patients show a hypomethylation of the IGF2/H19 imprinting region in 11p15, whereas the additional 10% carry a maternal uniparental disomy of chromosome 7. In single cases, cytogenetic aberrations can be detected. Nevertheless, there still remain 50% of SRS patients without known genetic/epigenetic alterations. To find out whether submicroscopic imbalances contribute to the aetiology of SRS, 20 idiopathic SRS patients were screened with the Affymetrix GeneChip Human Mapping 500 K array set. Apart from known apathogenic copy number variations, we identified one patient with a 12q14 microdeletion. The 12q14 microdeletion syndrome is characterised by dwarfism but it additionally includes mental retardation and osteopoikilosis. The deletion in our patient is smaller than those in the 12q14 microdeletion carriers but it also affects the LEMD3 and the HMGA2 genes. LEMD3 haploinsufficiency and point mutations have been previously associated with osteopoikilosis but radiographs of our patient at the age of 16 years did not reveal any hint for osteopoikilosis lesions. Haploinsufficiency of HMGA2 is probably responsible for aberrant growth in 12q14 microdeletion syndrome. However, in this study, a general role of HMGA2 mutations for SRS was excluded by sequencing of 20 idiopathic patients. In conclusion, our results exclude a common cryptic chromosomal imbalance in idiopathic SRS patients but show that chromosomal aberrations are relevant in this disease. Thus, molecular karyotyping is indicated in SRS and should be included in the diagnostic algorithm.

Genotype-phenotype studies in infantile spinal muscular atrophy (SMA) type I in Germany: implications for clinical trials and genetic counselling.

We reviewed the natural history and assessed the SMN2 copy number of 66 patients with infantile spinal muscular atrophy (SMA) type I born between 2000 and 2005 in Germany whose diagnosis was confirmed by a homozygous SMN1 deletion in the first 6 months of life. After excluding patients who had received valproic acid, the median/mean age at disease endpoint was 6.1/7.3 months (range 0.0-34.0). Four (6.1%) patients with one SMN2 copy had severe SMA type '0' with joint contractures and respiratory distress from birth. Median/mean age at onset (months) in 57 (86.3%) patients with two SMN2 copies was 1.2/1.3, and 3.5/3.4 in 5 (7.6%) patients with three SMN2 copies. Median/mean age at disease endpoint was 6.5/7.8 months (range 0.5-30) in patients with two SMN2 copies. All patients with three SMN2 copies were still alive at 10-55 months, two of them under permanent ventilation. Our data are relevant for prognostication and genetic counselling. The observed clinical variability, especially in the group with two SMN2 copies, might be important for clinical trials in SMA I where a possible control group could be defined as follows: age at onset within 4-5 months, age at genetic diagnosis <6 months, two SMN2 copies present, head control in less than 10%, no respiratory distress from birth, disease endpoint either age at death or age at permanent ventilation.

Genetic variation in schizophrenia-risk-gene dysbindin 1 modulates brain activation in anterior cingulate cortex and right temporal gyrus during language production in healthy individuals.

Genetic variation in dysbindin 1 (DTNBP1) gene region tagged by SNP rs1018381 exhibits a linkage with cognitive deficits in patients with schizophrenia and healthy subjects. Language production deficits are core features of schizophrenia with more impairment in semantic than lexical verbal fluency tasks. We investigated the link between brain activation and DTNBP1 SNP rs1018381 during semantic verbal fluency task in a German healthy population. 46 healthy subjects genotyped for SNP rs1018381 status were divided in heterozygous risk-allele carriers (T/C) and homozygous non-carriers (C/C). Neural correlates of semantic verbal fluency were investigated with functional magnetic resonance imaging (fMRI). Stronger right hemispherical brain activation in anterior cingulate gyrus (BA 24), superior (BA 22, 38) and middle (BA 21) temporal gyrus was observed in the carriers compared to non-carriers. Brain activations occurred in the absence of task performance differences. No significant correlations were found between personality traits and brain activation differences. The results point to an influence of genetic variation in DTNBP1 gene region tagged by SNP rs1018381 on neural correlates of language production. Carriers may exhibit higher processing efforts to reach the same behavioural performance as non-carriers as reflected in activation of schizophrenia-related regions.