Genetic studies of complement C4 in man

Summary A C4 variant found in about 5% of the population is described. The fast-moving part of this variant is governed by an allele (F ^x) codominant to F. The F ^x allele is in very strong linkage disequilibrium with HLA-B17 as the linkage disequilibrium parameter accounted for nearly 100% of the haplotype frequency of B17, F ^x. The strong association is also evidenced by the study of 11 families segregating for the F ^x allele. There was no instance of recombination between C4 and HLA in 36 informative meioses.     

HLA genotypes and HLA-linked genetic markers in Italian patients with classical 21-hydroxylase deficiency

Summary HLA genotype and HLA-linked marker data for 40 unrelated patients from central Italy and 2 unrelated patients from Sardinia with congenital adrenal hyperplasia due to 21-hydroxylase deficiency (21-OH-def) were analyzed. The results confirm that the HLA-linked 21-OH-def gene is associated with several different HLA determinants and complete HLA haplotypes, although the only determinant with significantly increased frequency was the complement C2 allele C2B. The HLA antigens B8 and DR3 were found in significantly decreased frequencies. The haplotype A3, Cw6, Bw47, BfF, DR7, which is exceptionally rare in the general population but which has been found in many other 21-OH-def patients from diverse geographical origins, was also found in one of the Italian patients. This and other HLA haplotype associations found among the Italian patients may represent mutations that have occurred on HLA haplotypes with genetic linkage disequilibrium or, alternatively, may represent mutations that have not yet had time to become randomly associated with different HLA complex determinants. The marked negative associations with B8 and DR3 could, however, result from an interaction between the gene products of the HLA complex and the 21-OH-def phenotype.     

A linkage study of hereditary ataxias and related disorders

Genetic markers controlled by 21 genetic systems were studied in 13 families containing members suffering from various hereditary disorders involving ataxia. Classical cerebellar ataxia was present in four, Friedreich ataxia in two, hereditary spastic paraplegia in four, and the Charcot-Marie-Tooth syndrome in three families. In each family, every available member above the lowest age at onset observed in that family, was subjected to a thorough clinical investigation and blood was sampled for investigation of genetic markers.The families with cerebellar ataxia and with Charcot-Marie-Tooth syndrome contained enough informative relatives to allow a formal linkage study using the lodscore method. Three of the pedigress with cerebellar ataxia gave evidence of linkage between the disease and the HLA system with a combined lodscore of 2.128 at a recombination fraction of 0.05 for both sexes combined. The recombination fraction was considerably higher in females than in males, although the difference between the two sexes was not statistically significant.Negative lodscores were obtained for the remaining family with cerebellar ataxia, which might be due to the fact that this family only provided information on recombination in females. However, the clinical features in this family differed from those in the other three families by a significantly higher frequency of dementia and pyramidal tract lesions. Based on these observations and on contradictory results in the literature concerning linkage between cerebellar ataxia and HLA, we suggest that there are two forms of cerebellar ataxia: One (CA¹) linked to HLA with symptoms restricted to lesions in the cerebellum and spinocerebellar system and another (CA²) not linked to HLA with symptoms from more wide-spread lesions of the CNS.None of the other genetic markers (except perhaps GLO) showed linkage to the cerebellar ataxias. Negative lodscores throughout with all 21 genetic markers were found in the families with Charcot-Marie-Tooth syndrome.There was no evidence for linkage between HLA on the one hand and Friedreich ataxia or hereditary spastic paraplegia on the other.List of Abbreviations HA Hereditary ataxias - HLA Major histocompatibility system - CA Cerebellar ataxia - FA Friedreich ataxia - HSP Hereditary spastic paraplegia - CMT Charcot-Marie-Tooth syndrome - MS Multiple sclerosis - Hp Haptoglobin - Gc Group-specific component - PGM Phosphoglucomutase, locus 1 - SP (AcP) Acid phosphatase - AK Adenylatekinase - PGD 6-phosphogluconatedehydrogenase - ADA Adenosinedeaminase - GPT Glutamate pyruvat transaminase - GT Galaktose-1-phosphat uridylyltransferase - EsP Carboxylesterase D - GLO Glyoxylase I This study was aided by grants from Warwara Larsen's Foundation, the Danish Multiple Sclerosis Society and the Medical Research Council     

Immunofixation for C2 typing: C2 allotypes in Spaniards in relation to HLA,Bf and C4

Summary C2 typing is performed by immunofixation with anti-C2 antiserum instead of by a hemolytic overlay. This method gives sharp band definition, is less cumbersome than the hemolytic overlay, gel files are easily made, and it also enables one to describe putative new nonhemolytic variants. C2 allele frequencies were studied in a sample of the normal Spanish population and were found to be similar to other Caucasoids. HLA-Bw62,-Cw3, and-DR4 were significantly associated with C2 B. Concordantly, the only C2^*B extended HLA haplotype found in family material was Bw62-Cw3-Bw6-(DR4)-Bf^*S-C2^*B-C4A^*3 B^*2-(GLO^*1). C4A^*4 B^*2 and C4A^*4 B^*4 are not found within the same haplotype together with C2^*B and Bw62 or Bw22 respectively, nor do other C2^*B haplotypes occur with common HLA-B alleles. These results may favour the hypothesis that the Bw62-C2^*B haplotype is produced by one mutation arising in the Bw62-C2^*C haplotype and that subsequent crossovers can explain other C2^*B haplotypes (including Bw22-C2^*B).     

Ancestral association between HLA and HFE H63D and C282Y gene mutations from northwest Colombia

A significant association between HFE gene mutations and the HLA-A*03-B*07 and HLA-A*29-B*44 haplotypes has been reported in the Spanish population. It has been proposed that these mutations are probably connected with Celtic and North African ancestry, respectively. We aimed to find the possible ancestral association between HLA alleles and haplotypes associated with the HFE gene (C282Y and H63D) mutations in 214 subjects from Antioquia, Colombia. These were 18 individuals with presumed hereditary hemochromatosis (“HH”) and 196 controls. The HLA-B*07 allele was in linkage disequilibrium (LD) with C282Y, while HLA-A*23, A*29, HLA-B*44, and B*49 were in LD with H63D. Altogether, our results show that, although the H63D mutation is more common in the Antioquia population, it is not associated with any particular HLA haplotype, whereas the C282Y mutation is associated with HLA-A*03-B*07, this supporting a northern Spaniard ancestry.     

A linkage map of mouse Chromosome 1 using an interspecific cross segregating for the gld autoimmunity mutation

An interspecific backross was used to define a high resolution linkage map of mouse Chromosome (Chr) 1 and to analyze the segregation of the generalized lymphoproliferative disease (gld) mutation. Mice homozygous for gld have multiple features of autoimmune disease. Analysis of up to 428 progeny from the backcross [(C3H/HeJ-gld x Mus spretus)F₁ x C3H/HeJ-gld] established a map that spans 77.6 cM and includes 56 markers distributed over 34 ordered genetic loci. The gld mutation was mapped to a less than 1 cM segment on distal mouse Chr 1 using 357 gld phenotype-positive backcross mice. A second backcross, between the laboratory strains C57BL/6J and SWR/J, was examined to compare recombination frequency between selected markers on mouse Chr 1. Significant differences in crossover frequency were demonstrated between the interspecific backcross and the inbred laboratory cross for the entire interval studied. Sex difference in meiotic crossover frequency was also significant in the laboratory mouse cross. Two linkage groups known to be conserved between segments of mouse Chr 1 and the long arm of human Chrs 1 and 2 where further defined and a new conserved linkage group was identified that includes markers of distal mouse Chr 1 and human Chr 1, bands q32 to q42.     

The genetics of aryl hydrocarbon hydroxylase induction in mice: A single gene difference between C57BL/6J and DBA/2J

Twenty-one inbred strains of mice were surveyed for inducibility of hepatic aryl hydrocarbon hydroxylase (AHH) activity by the carcinogen 3-methylcholanthrene (MC). In 11 strains given MC, AHH activity increased 1.3- to 5-fold (inducible), whereas ten strains responded with a less than 0.5-fold increase (noninducible). Neither the inducible nor the noninducible class was homogeneous, and in each considerable variation was found in both the basal activity of AHH and the response to MC. Strains DBA/2J and C57BL/6J were chosen to represent the noninducible and inducible classes, respectively. In the crosses (C57BL/6 × DBA/2)F₁ × DBA/2 and (C57BL/6 × DBA/2)F₂, inducibility segregated as a single autosomal dominant gene. The gene symbols Ahh ⁱ and Ahh ⁿ are proposed for the alleles present in C57BL/6J and DBA/2J, respectively. No genetic linkage was found between the Ahh locus and the following loci: b, d, Es-1, Es-3, Gpd-1, Hbb, Id-1, Pgm-1, and sex. Some implications of this work in the study of mammalian enzyme induction and chemically induced carcinogenesis are discussed. There is a positive correlation between AHH inducibility and the development of an inflammatory response to the topical application of the carcinogen 7,12-dimethylbenzanthracene.     

Family studies of complement C4 and HLA in man

Summary At least 12 different C4 gene products with a three band pattern have been identified after electrophoresis of sera pretreated with neuraminidase.Segregation analysis showed at least 12 different C4 haplotypes (or supergenes), of which five represent a single gene product and seven are duplications each composed of an F and an S gene. The data analyzed with respect to linkage showed one recombinant between the C4 and the HLAB loci in 154 meioses giving a map distance of C4 HLAB of 0.6 cM. Another recombinant between the C4 and the HLAD loci was found in 101 meioses giving a map distance of C4 HLAD of 1.0 cM.Linkage disequilibrium was found between at least eight C4 haplotypes and certain alleles at the HLAB as well as the HLAD loci. Examinations of 15 families selected through a proband with HLAA 25, HLAB 18 and C2 ^* Q0 showed that in almost all cases a slight variant of the C4 supergene F3S2 followed the haplotype HLAA ^* 25 HLAB ^* 18 C2 ^* Q0. No associations were found between the two duplications C4F ^* 3 C4S ^* 2 and C4F ^* 3 C4S ^* 1 and the HLA loci. These findings may indicate that these C4 haplotypes were the original ones preceding the other C4 haplotypes.     

Chronic cerebrospinal vascular insufficiency is not associated with HLA DRB1*1501 status in multiple sclerosis patients

Background

Chronic cerebrospinal venous insufficiency (CCSVI) was described as a vascular condition characterized by anomalies of veins outside the skull was reported to be associated with multiple sclerosis (MS). The objective was to assess the associations between HLA DRB1*1501 status and the occurrence of CCSVI in MS patients.

Methodology/Principal Findings

This study included 423 of 499 subjects enrolled in the Combined Transcranial and Extracranial Venous Doppler Evaluation (CTEVD) study. The HLA DRB1*1501 status was obtained in 268 MS patients and 155 controls by genotyping rs3135005, a SNP associated with DRB1*1501 status. All subjects underwent a clinical examination and Doppler scan of the head and neck. The frequency of CCSVI was higher (OR = 4.52, p<0.001) in the MS group 56.0% vs. 21.9% in the controls group and also higher in the progressive MS group 69.8% vs. 49.5% in the non-progressive MS group. The 51.9% frequency of HLA DRB1*1501 positivity (HLA⁺) in MS was higher compared (OR = 2.33, p<0.001) to 31.6% to controls. The HLA⁺ frequency in the non-progressive (51.6%) and progressive MS groups (52.3%) was similar. The frequency of HLA⁺ CCSVI⁺ was 40.7% in progressive MS, 27.5% in non-progressive MS and 8.4% in controls. The presence of CCSVI was independent of HLA DRB1*1501 status in MS patients.

Conclusions/Significance

The lack of strong associations of CCSVI with HLA DRB1*1501 suggests that the role of the underlying associations of CCSVI in MS should be interpreted with caution. Further longitudinal studies should determine whether interactions between these factors can contribute to disease progression in MS.     

Mapping of a quantitative trait locus for morphine withdrawal severity

Chronic morphine exposure results in physical dependence, manifested by physical symptoms during naloxone-precipitated withdrawal. Jumping frequency is widely considered the most sensitive and reliable index of withdrawal intensity in mice. Inbred mouse strains surveyed for naloxone-precipitated withdrawal display large and significant strain differences in jumping frequency, including an approximately tenfold difference between C57BL/6 and 129P3 mice. In the present study, (B6 × 129)F₂ hybrid mice were given daily morphine injections for four days using an escalating dosing schedule, and naloxone-precipitated withdrawal on day 5 was measured. A full-genome scan for linkage to phenotypic data was performed using polymorphic microsatellite markers. Significant linkage was observed between withdrawal jumping frequencies and a 28 cM-wide region of Chromosome 1 (32–60 cM; peak at 51 cM), accounting for 20% of the overall phenotypic variance. Two other suggestive QTLs were found, on Chromosomes 5 and 10, and an additive model fitting all three loci accounted for 43% of the total variance. F₂ mice were also assessed for changes in morphine analgesic potency using the tail-withdrawal test in dose–response studies on days 1 and 4. No linkage was observed between Chromosomes 1, 5, and 10 and morphine analgesic tolerance, suggestive of genetic dissociation of naloxone-precipitated withdrawal from morphine and chronic morphine intake per se. The significant quantitative trait locus for naloxone-precipitated withdrawal severity in morphine-dependent mice, which we name Depmq1, may prove to be of considerable heuristic value once the underlying gene or genes are identified.     

Sensitivity to dietary obesity linked to a locus on Chromosome 15 in a CAST/Ei × C57BL/6J F2 intercross

Details of a new model of diet-dependent polygenic obesity are presented. CAST/Ei (Mus m. castaneus) mice remain lean after 12 weeks on a high-fat (32 kcal% fat) diet, while C57BL/6J mice become obese. The genes responsible for the obesity segregate in an F₂ population derived from an intercross between CAST/Ei and C57BL/6J mice. Quantitative trait analysis, with simple sequence length polymorphisms (SSLPs) at loci previously linked to rodent obesities, identified a quantitative trait locus (QTL) on Chromosome (Chr) 15, accounting for approximately 9% of the variance in adiposity and 14% of the variance in mesenteric depot size. This locus appears to be at the same location as the dietary obesity-3 (Do3) locus controlling body fat content, which was previously identified in an F₂ population derived from an SWR/J × AKR/J cross. This is also at the same location as the multigenic obesity-4 (Mob4) locus found in BSB mice, which display spontaneous polygenic obesity. Suggestive linkage also was found at loci close to the single gene mutations A ^y on Chr 2 and tub on Chr 7. Received 15 January 1996 / Accepted 12 May 1996     

Major histocompatibility complex class I associations in iron overload: evidence for a new link between the HFE H63D mutation, HLA-A29, and non-classical forms of hemochromatosis

 The present study is an analysis of the frequencies of HFE mutations in patients with different forms of iron overload compared with the frequencies found in healthy subjects from the same region. The frequencies of HLA-A and -B antigens and HLA haplotypes were also analyzed in the same subjects. The study population included: 71 healthy individuals; 39 genetically and clinically well-characterized patients with genetic hemochromatosis (HH); and 25 patients with non-classical forms of iron overload (NCH), excluding secondary hemochromatosis. All subjects were HLA-typed and HFE-genotyped by the oligonucleotide ligation assay (OLA). The gene frequencies found for the C282Y and H63D mutations of HFE were respectively: 0.03 and 0.23 in healthy individuals, 0.86 and 0.04 in HH patients, and 0.08 and 0.48 in NCH patients. An expected significant association between HH and HLA-A3 was observed, which was found to be in linkage disequilibrium with the C282Y mutation. A new association was seen, however, between HLA-A29 and NCH, in linkage disequilibrium with the H63D mutation. Again as expected, the HLA-B antigen B7 was associated with HH in linkage disequilibrium with HLA-A3. In addition, the HLA-B antigen B44 was found to be associated with NCH but not in linkage disequilibrium with either A29 or the H63D mutation. In conclusion, a new association of the HFE H63D mutation with forms of hemochromatosis other than HH and a new association between the HLA phenotype A29 and the HFE H63D mutation were found in the same patients. These findings reinforce evidence for the involvement of the major histocompatibility class I in iron metabolism, supporting the notion of a physiological role for the immunological system in the regulation of iron load. Received: 11 June 1997 / Revised: 29 October 1997     

High Resolution Human Leukocyte Antigen Class I Allele Frequencies and HIV-1 Infection Associations in Chinese Han and Uyghur Cohorts

Background

Host immunogenetic factors such as HLA class I polymorphism are important to HIV-1 infection risk and AIDS progression. Previous studies using high-resolution HLA class I profile data of Chinese populations appeared insufficient to provide information for HIV-1 vaccine development and clinical trial design. Here we reported HLA class I association with HIV-1 susceptibility in a Chinese Han and a Chinese Uyghur cohort.

Methodology/Principal Findings

Our cohort included 327 Han and 161 Uyghur ethnic individuals. Each cohort included HIV-1 seropositive and HIV-1 seronegative subjects. Four-digit HLA class I typing was performed by sequencing-based typing and high-resolution PCR-sequence specific primer. We compared the HLA class I allele and inferred haplotype frequencies between HIV-1 seropositive and seronegative groups. A neighbor-joining tree between our cohorts and other populations was constructed based on allele frequencies of HLA-A and HLA-B loci. We identified 58 HLA-A, 75 HLA-B, and 32 HLA-Cw distinct alleles from our cohort and no novel alleles. The frequency of HLA-B*5201 and A*0301 was significantly higher in the Han HIV-1 negative group. The frequency of HLA-B*5101 was significantly higher in the Uyghur HIV-1 negative group. We observed statistically significant increases in expectation-maximization (EM) algorithm predicted haplotype frequencies of HLA-A*0201-B*5101 in the Uyghur HIV-1 negative group, and of Cw*0304-B*4001 in the Han HIV-1 negative group. The B62s supertype frequency was found to be significantly higher in the Han HIV-1 negative group than in the Han HIV-1 positive group.

Conclusions

At the four-digit level, several HLA class I alleles and haplotypes were associated with lower HIV-1 susceptibility. Homogeneity of HLA class I and Bw4/Bw6 heterozygosity were not associated with HIV-1 susceptibility in our cohort. These observations contribute to the Chinese HLA database and could prove useful in the development of HIV-1 vaccine candidates.     

Genotypic diversity of the Killer Cell Immunoglobulin-like Receptors (KIR) and their HLA class I Ligands in a Saudi population

Killer Cell Immunoglobulin-like Receptors (KIR) have been used as good markers for the study of genetic predisposition in many diseases and in human genetic population dynamics. In this context, we have investigated the genetic diversity of KIR genes and their main HLA class I ligands in Saudi population and compared the data with other studies of neighboring populations. One hundred and fourteen randomly selected healthy Saudi subjects were genotyped for the presence or absence of 16 KIR genes and their HLA-C1, -C2, -Bw4^Thr80 and Bw4^Ile80 groups, using a PCR-SSP technique. The results show the occurrence of the framework genes (3DL2, 3DL3 and 2DL4) and the pseudogenes (2DP1 and 3DP1) at highest frequencies. All inhibitory KIR (iKIR) genes appeared at higher frequencies than activating genes (aKIR), except for 2DS4 with a frequency of 90.35%. A total of 55 different genotypes were observed appearing at different frequencies, where 12 are considered novel. Two haplotypes were characterized, AA and Bx (BB and AB), which were observed in 24.5% and 75.5% respectively of the studied group. The frequencies of iKIR + HLA associations were found to be much higher than aKIR + HLA. KIR genes frequencies in the Saudi population are comparable with other Middle Eastern and North African populations.     

Mutant allele frequencies in domestic cat populations on the Spanish Mediterranean coast,and their genetic distances from other European and North African cat populations

New genetic profiles of domestic cat populations were studied in different parts of the Spanish Mediterranean coast. The t ^b frequency is the most outstanding differentiated factor between the populations analyzed. The low t ^b frequencies found in Barcelona and other Catalan localities are the most important characteristic of these populations and, probably, find an explanation in the extraordinarily high historical and commercial relationships between Barcelona and the Eastern Mediterranean (Greece, especially) during the 14th and 15th centuries. In other Spanish Mediterranean littoral cities the t ^b frequencies reach higher values and are more similar to those found in other Western European cat populations. Significant differences found in certain allelic frequencies (Orange and Blotched) caused by the urban-rural effect are discovered between Barcelona and two nearby rural cat populations.     

Human phosphoglycolate phosphatase (PGP) E.C.3.1.3.18: Linkage analysis

Summary Linkage data on phosphoglycolate phosphatase (PGP) E.C.3.1.3.18 and 26 other human genetic markers are presented. One hundred and one families from the southwestern area of Germany were tested. Close linkage between PGP and the following markers could be ruled out: AB0, acP, ADA, GPT, PGM₁, GLO, HLA, and PGM₃. There is some evidence for possible linkage with MNSs, Rh, Gm and EsD. Family segregation data confirm the hypothesis formerly established by Barker and Hopkinson: three common alleles PGP¹, PGP² and PGP³ at an autosomal locus PGP.Supported by the Deutsche ForschungsgemeinschaftSupported by DAAD and Portuguese Inst. for Scientific Research (INIC)     

The genetic control of natural killer cell activity and its association with in vivo resistance against a moloney lymphoma isograft

Spleens of normal young mice of certain strains contain lymphocytes that can kill strain A-derived YAC-1 lymphoma cells in a⁵¹Cr release cytotoxic assay in vitro. We have previously classified mouse genotypes as high or low reactors, according to their responses in this test. In vivo resistance to small numbers of YAC ascites lymphoma cells is correlated with in vitro cytolytic activity. In vitro and in vivo tests were carried out on the same individual (A x C57BL)F₁ x A backcross mice. Natural in vitro killer cell activity appeared to be under polygenic control, including a strong H-2-linked factor. No linkage was found with five different isozyme loci, with theIg-l locus or with C5 serum activity. Also in vivo resistance showed strong linkage with theH-2 complex. In (A x CBA)F₁ x A backcross mice, a weak linkage was found with the coat color locusC. There was a correlation between in vitro killer activity and in vivo resistance in the same backcross mice. In vivo resistance was particularly strong in mice that combined theH-2 ^b -linked resistance factor with a high cytolytic activity in vitro.     

Genetics of obesity in KK mouse and effects of A y allele on quantitative regulation

KK mouse is known as a polygenic model for non-insulin-dependent diabetes mellitus with moderate obesity. To identify the quantitative trait loci (QTLs) responsible for the body weight in KK, linkage analysis with 97 microsatellite markers was carried out into 192 F₂ progeny, comprising 93 mice with a/a genotype at agouti locus and 99 mice with A ^y /a genotype, of a cross between C57BL/6J female and KK-A^y (A^y congenic) male, thereby the influence of A ^y allele on the quantitative regulation of body weight was also examined. In F₂ a/a mice, we identified a QTL on Chromosome (Chr) 4, and two loci with suggestive linkage on Chrs 15 and 18. In F₂ A ^y /a mice, a QTL was identified on Chr 6, and two loci with suggestive linkage were identified on Chrs 4 and 16. That the QTL on Chr 4 was held in common between F₂ a/a and F₂ A ^y /a progenies implies that this locus may be a primary component regulating body weight in KK and KK-A^y. These results suggest that the body weight in KK is controlled by multiple genes, and the different combination of loci is involved in the presence of A ^y allele. The QTL on Chr 6 seemed to determine the body weight by controlling fat deposition, because the linkage was identified on body weight and adiposity, and is suggested to be a component involved in the metabolic pathway in obesity caused by the A ^y allele. Received: 16 December 1997 / Accepted: 16 March 1998     

Genetic linkage relations of the sixth component of complement (C6)

Summary Linkage relations between the C6 and 33 other genetic marker loci have been analyzed in Norwegian pedigrees, including 114 matings with 388 informative children, by use of the MOSM computer program. No suggestion of linkage was found. Very close or close linkage (<0.06) has been ruled out for males between C6 and the following 19 marker loci: GPT, HLA+Bf, Rh, C3, Hp, PGM ₃, Km, Gm, Fy, Gc, AB0, Jk, GLO ₁, K, MNSs, PTC, ACP ₁, PGM ₁ and Pi. For several of the relations even loose linkage is unlikely.     

Genetic variation of an acid phosphatase (Acp-2) in the laboratory rat: Possible homology with mouse AP-1 and human ACP2

A genetic locus controlling the electrophoretic mobility of an acid phosphatase in the rat (Rattus norvegicus) is described. The locus, designed Acp-2, is not expressed in erythrocytes but is expressed in all other tissues studied. The product of Acp-2 hydrolyzes a wide variety of phosphate monoesters and is inhibited by l(+)-tartaric acid. Inbred rat strains have fixed either allele Acp-2^a or allele Acp-2^b. Codominant expression is observed in the respective F₁ hybrids. Backcross progenies revealed the expected 1:1 segregation ratio. Possible loose linkage was found between the Acp-2 and the Pep-3 gene loci at a recombination frequency of 0.36±0.06.Supported by the Deutsche Forschungsgemeinschaft (Grant Be 352/15) and by a grant from the Alexander-von-Humboldt-Stiftung (VB2-FLF).