Evidence for anticipation in schizophrenia.

Anticipation, or increasing severity of a disorder across successive generations, is a genetic phenomenon with an identified molecular mechanism: expansion of unstable trinucleotide repeat sequences. This study examined anticipation in familial schizophrenia. Three generations of siblines from the affected side of families selected for unilineal, autosomal dominant-like inheritance of schizophrenia were studied (n = 186). Across generations more subjects were hospitalized with psychotic illness (P < .001), at progressively earlier ages (P < .0001), and with increasing severity of illness (P < .0003). The results indicate that anticipation is present in familial schizophrenia. These findings support both an active search for unstable trinucleotide repeat sequences in schizophrenia and reconsideration of the genetic model used for linkage studies in this disorder.     

Non-satellite repetitive human DNA families. Sequence properties and evidence for occurrence in chimpanzee DNA

Repetitive human DNA, fractionated on CsCl gradients following hydroxyapatite isolation, contains two complex DNA fractions, the 1.703 and 1.714 DNA families (Marx, K.A., Allen, J.R. and Hearst, J.E. (1976) Biochim. Biophys. Acta 425, 129-147). Biphasic Topt profiles, obtained in DNA excess hybridizations with cRNA tracers from each DNA family, have been shown to be the likely result of a fast kinetic component hybridizing at higher temperatures (67 degrees C peak) and this fast plus a slow kinetic component both hybridizing at lower temperatures (47 degrees C peak). Equilibrium CsCl gradient DNA-cRNA hybrid distributions support previous interpretations of the sequence composition of both DNA families. That is, the fast component is a relatively undiverged repetitive sequence of recent origin, while the slow component is a highly diverged, less thermally stabile, old primate sequence. This interpretation is further strengthened by cRNA tracer hybridization experiments in chimpanzee DNA excess where the fast component appears to be absent and the slow component present.     

Search for retroviral related DNA polymorphisms using RAPD PCR in schizophrenia

Random amplification of polymorphic DNA (RAPD) is widely used to detect polymorphisms in many organisms. Individual (or strain) specific amplified bands are generated with single or pairs of primers in PCR reactions and can serve as genetic markers. We have used this method to generate a large number of reproducible bands with single primers, random and retroviral related, on 92 human DNA samples. Theoretically, RAPD PCR presents a logical approach for assessing variability among individuals. We used ten retroviral related primers (12, 20 and 22 bp) and eight random primers (10 bp) to assess individual differences in the context of testing the retroviral hypothesis for schizophrenia. Three pairs of discordant monozygotic twins, four pairs of discordant full sibs and 53 schizophrenic individuals with 25 of their unrelated matched controls were analyzed. Ten of these primers resulted in a total of approx. 850 amplified bands (65-110 bands per primer). Almost all of these bands were identical among each individual analyzed. However, the results are inconclusive with respect to the retroviral hypothesis for schizophrenia. The general lack of RAPD polymorphism in this study may argue for mechanisms other than rearrangements such as inversions, associated with the evolution of the human genome.     

Evidence for genetic anticipation in hereditary non-polyposis colorectal cancer

Hereditary non-polyposis colorectal cancer (HNPCC) is an autosomal, dominantly inherited, colorectal cancer (CRC) predisposition syndrome caused by germline mutations in DNA mismatch repair (MMR) genes, predominantly MLH1 and MSH2. Thus far, only limited data exist on the occurrence of genetic anticipation in HNPCC, i.e. the earlier age at diagnosis of CRC in successive generations. Performing nonparametric distribution-free statistical analyses, we investigated 55 parent–child pairs who had been diagnosed with CRC and who came from 21 Swiss HNPCC families with characterised MMR germline mutation (15 in MLH1 and 6 in MSH2). The overall median age at diagnosis was 43 years, with an interquartile range (IQR) of 14 and incidence ages ranging from 18 to 62 years. Descendants of HNPCC patients (median age at diagnosis 39 years, IQR=12) were found to be diagnosed with CRC significantly earlier than their parents (47 years, IQR=10), with the median of the paired age difference amounting to 8 years (IQR=15; P<0.0001). Birth cohort effects could be excluded, since the same, statistically significant, age difference was also observed in the oldest offspring birth cohort (birth year <1916; P=0.01). Genetic anticipation appeared to be more pronounced when the disease allele was transmitted through the father than through the mother (median age difference 11 vs. 4 years, respectively; both P<0.01). If confirmed in larger, ideally prospective studies, these results may have important implications for genetic counselling and clinical management of HNPCC families.     

Search for an adenine photoproduct in DNA.

Poly(d[14C]A), p(dA)2, and [14C]adenosine-labeled DNA were irradiated at 254 nm with fluences up to 50 J/m2, and then following formic acid hydrolysis at 170 degrees C WERE SUBJECTED TO PAPER CHROMAtography using a butanol:water:acetic acid (80:30:12) solvent system. For poly(dA), up to 25% of the radioactivity appeared as fluorescent material located in the Rf 0.21-0.29 region. The hydrolysate of the purified photoproduct, p(dA)2, isolated from irradiated p(dA)2 by DEAE chromatography also had an Rf of 0.29 as well as an absorbance maximum at 310 nm. In all cases studied, however, the photoproduct yield in the Rf 0.29 region for native DNA was less than 2%. Denaturation of the DNA appeared to enhance the yield slightly, although no pronounced peak in this region of the chromatogram was discerned. Mechanistic studies indicate that the yield of the adenine photoproduct in poly(dA) is favored by base stacking, has a singlet excimer as a precursor, and is quenched by hydrogen bonding to a pyrimidine. It is concluded that the yield of the adenine photoproduct in both native and denatured DNA is considerably less than in poly (dA) and in all probability does not represent a biologically significant product.     

Microsatellite DNA evidence for genetic drift and philopatry in Svalbard reindeer

Mainland populations of Arctic reindeer and caribou Rangifer tarandus often undergo extensive movements, whereas populations on islands tend to be isolated and sedentary. To characterize the genetic consequences of this difference, levels of genetic diversity and subdivision of Svalbard reindeer (R. t. platyrhynchus) from two adjacent areas on Nordenskjiöldland, Spitsbergen were estimated using data from up to 14 microsatellites. The mean number of alleles per locus in Svalbard reindeer was 2.4 and mean expected heterozygosity per locus was 0.36. The latter value was significantly lower than in Canadian caribou and Norwegian reindeer but higher than in some other cervid species. Large samples of females (n = 743) and small samples of males (n = 38) from two sites ≈ 45 km apart showed genetic subdivision, which could be due to local population fluctuations or limited gene flow. To our knowledge, this is the first study to report significant differentiation at microsatellite loci in Rangifer at such short geographical distances. Neither population showed genetic evidence for recent population bottlenecks when loci unbiased with respect to heterozygosity were analysed. In contrast, false signals of a recent bottleneck were detected when loci upwardly biased with respect to heterozygosity were analysed. Thus, Svalbard reindeer appeared to conform to the paradigm of island populations made genetically depauperate by genetic drift.     

Molecular genetic analysis of two families with keratosis follicularis spinulosa decalvans: refinement of gene localization and evidence for genetic heterogeneity

X-linked keratosis follicularis spinulosa decalvans (KFSD) is a rare disorder affecting both skin and eyes. In the two extended KFSD families analysed to date, the gene was mapped to Xp22.13–p22.2. By analyzing several new markers in this region, we were able to narrow the candidate region to a 1-Mb interval between DXS7161 and (DXS7593, DXS7105) in the large Dutch pedigree. In addition, we analyzed 23 markers in Xp21.2– p22.2 in a German family with KFSD. Haplotype and recombination analysis positioned the KFSD gene in this family most likely outside the candidate region on Xp22.13–p22.2. This finding is suggestive for genetic heterogeneity: in this pedigree there is either another locus on the X-chromosome, or KFSD is transmitted here as an autosomal dominant trait with variable expression. Received: 28 May 1996 / Accepted: 28 May 1997     

Molecular and clinical study of 18 families with ADCA type II: evidence for genetic heterogeneity and de novo mutation.

The SCA7 mutation has been found in 54 patients and 7 at-risk subjects from 17 families who have autosomal dominant cerebellar ataxia (ADCA) II with progressive pigmentary maculopathy. In one isolated case, haplotype reconstruction through three generations confirmed a de novo mutation owing to paternal meiotic instability. Different disease-associated haplotypes segregated among the SCA7-positive kindreds, which indicated a multiple origin of the mutation. One family with the clinical phenotype of ADCA type II did not have the CAG expansion that indicated locus heterogeneity. The distribution of the repeat size in 944 independent normal chromosomes from controls, unaffected at-risk subjects, and one affected individual fell into two ranges. The majority of the alleles were in the first range of 7-19 CAG repeats. A second range could be identified with 28-35 repeats, and we provide evidence that these repeats represent intermediate alleles that are prone to further expansion. The repeat size of the pathological allele, the widest reported for all CAG-repeat disorders, ranged from 37 to approximately 220. The repeat size showed significant negative correlation with both age at onset and age at death. Analysis of the clinical features in the patients with SCA7 confirmed that the most frequently associated features are pigmentary maculopathy, pyramidal tract involvement, and slow saccades. The subjects with <49 repeats tended to have a less complicated neurological phenotype and a longer disease duration, whereas the converse applied to subjects with >/=49 repeats. The degree of instability during meiotic transmission was greater than in all other CAG-repeat disorders and was particularly striking in paternal transmission, in which a median increase in repeat size of 6 and an interquartile range of 12 were observed, versus a median increase of 3 and interquartile range of 3.5 in maternal transmission.     

Search for a DNA gyrase in mammalian mitochondria.

Incorporation of labeled deoxynucleoside triphosphates into mtDNA by isolated rat liver mitochondria has been shown previously to reflect DNA replication. We have used this system to seek evidence for a mtDNA gyrase. Coumermycin, novobiocin, nalidixic acid, and oxolinic acid are known to be inhibitors of Escherichia coli gyrase, to inhibit E. coli DNA replication, to abolish colicin E1 replication, and to depress the supercoiling of phage lambda DNA, the last two via inhibition of the DNA gyrase. Our results show that these agents inhibit [3H]dATP incorporation into bulk mtDNA at concentrations similar to those used for E. coli. Analysis by sucrose gradient sedimentation confirms the inhibition and shows further that the synthesis of the highly supercoiled form of mtDNA (i.e. 39 S DNA) is depressed relative to other mtDNA forms (i.e. 27 S DNA), suggesting an inhibition of the supercoiling process. Analysis of the DNA by CsCl/propidium diiodide centrifugation shows, in addition, that incubation with coumermycin results in the appearance of a mtDNA form shown to be relaxed mtDNA. The results are consistent with the occurrence of a mtDNA gyrase and its operation in mtDNA replication.     

Additional glomangioma families link to chromosome 1p: no evidence for genetic heterogeneity

Venous malformations are a common abnormality of the vasculature that may occur sporadically or, more rarely, as an autosomal dominant trait. One familial form of venous malformations has previously been linked to chromosome 9p. Mutations in the gene encoding Tie2, an endothelial specific receptor tyrosine kinase, have been identified in four different families. Glomangiomas are a subtype of venous malformations with glomus cell involvement. These cutaneous lesions can be inherited as an autosomal dominant disease with reduced penetrance and variable expressivity. We present evidence of linkage to chromosome 1p21-1p22 using four new glomangioma families, with a combined maximum two-point lod score of 7.32 at marker D1S2804. Markers D1S2129 and D1S2881 define the 24-cM linkage interval determined by recombination within affected individuals. A recent report also showed linkage of the glomangioma locus to chromosome 1p. A total of 9 families now map to this region, suggesting a decreased likelihood of locus heterogenity in familial glomangiomas. Investigation of candidate genes within the interval should provide new insights into lesion formation in inherited venous malformations.     

Support for association of schizophrenia with genetic variation in the 6p22.3 gene,dysbindin, in sib-pair families with linkage and in an additional sample of triad families

Genetic variants in a gene on 6p22.3, dysbindin, have been shown recently to be associated with schizophrenia (Straub et al. 2002a). There is no doubt that replication in other independent samples would enhance the significance of this finding considerably. Since the gene is located in the center of the linkage peak on chromosome 6p that we reported earlier, we decided to test six of the most positive DNA polymorphisms in a sib-pair sample and in an independently ascertained sample of triads comprising 203 families, including the families for which we detected linkage on chromosome 6p. Evidence for association was observed in the two samples separately as well as in the combined sample (P=.00068 for SNP rs760761). Multilocus haplotype analysis increased the significance further to .00002 for a two-locus haplotype and to .00001 for a three-locus haplotype. Estimation of frequencies for six-locus haplotypes revealed one common haplotype with a frequency of 73.4% in transmitted, and only 57.6% in nontransmitted, parental haplotypes. All other six-locus haplotypes occurring at a frequency of >1% were less often transmitted than nontransmitted. Our results represent a first successful replication of linkage disequilibrium in psychiatric genetics detected in a region with previous evidence of linkage and will encourage the search for causes of schizophrenia by the genetic approach.     

Complementation and hybridization evidence for additional families of type I DNA restriction and modification genes in Salmonella serotypes.

Of eight Salmonella, serB-linked hsd genes for the restriction and modification of DNA transferred to Escherichia coli/Salmonella hybrids, only two--those with SM and ST (S. muenchen and S. thompson, respectively) specificities--may have weakly complemented rSB- and none complemented rK-. An A-specific DNA probe failed to hybridize to HindIII-restricted fragments of each of the hybrids, but an SB (S. typhimurium)-specific probe hybridized to DNA from the hybrid with ST specificity. These results indicate that additional families of the type I hsd genes may exist.     

No evidence of expansion of CAG or GAA repeats in schizophrenia families and monozygotic twins

Many diseases caused by trinucleotide expansion exhibit increased severity and decreased age of onset (genetic anticipation) in successive generations. Apparent evidence of genetic anticipation in schizophrenia has led to a search for trinucleotide repeat expansions. We have used several techniques, including Southern blot hybridization, repeat expansion detection (RED) and locus-specific PCR to search for expanded CAG/CTG repeats in 12 families from the United Kingdom and 11 from Iceland that are multiplex for schizophrenia and demonstrate anticipation. The unstable DNA theory could also explain discordance of phenotype for schizophrenia in pairs of monozygotic twins, where the affected twin has a greater number of repeats than the unaffected twin. We used these techniques to look for evidence of different CAG/CTG repeat size in 27 pairs of monozygotic twins who are either concordant or discordant for schizophrenia. We have found no evidence of an increase in CAG/CTG repeat size for affected members in the families, or for the affected twins in the MZ twin sample. Southern hybridization and RED analysis were also performed for the twin and family samples to look for evidence of expansion of GAA/TTC repeats. However, no evidence of expansion was found in either sample. Whilst these results suggest that these repeats are not involved in the etiology of schizophrenia, the techniques used for detecting repeat expansions have limits to their sensitivity. The involvement of other trinucleotide repeats or other expandable repeat sequences cannot be ruled out. Received: 8 September 1997 / Accepted: 13 March 1998     

Homozygosity mapping in families with Joubert syndrome identifies a locus on chromosome 9q34.3 and evidence for genetic heterogeneity

Joubert syndrome is a rare developmental defect of the cerebellar vermis, with autosomal recessive inheritance. The phenotype is highly variable and may include episodic hyperpnea, abnormal eye movements, hypotonia, ataxia, developmental delay, and mental retardation. Even within sibships the phenotype may vary, making it difficult to establish the exact clinical diagnostic boundaries of Joubert syndrome. To genetically localize the gene region, we have performed a whole-genome scan in two consanguineous families of Arabian/Iranian origins, with multiple affected probands. In one family, we detected linkage to the telomeric region of chromosome 9q, close to the marker D9S158, with a multipoint LOD score of Z=+3.7. The second family did not show linkage to this region, giving a first indication of genetic heterogeneity underlying Joubert syndrome. These findings were supported by subsequent analysis of two smaller families-one compatible with linkage to 9q; the other, unlinked. We conclude that Joubert syndrome is clinically and genetically heterogeneous and that one locus maps to chromosome 9q.     

High proportion of new mutations and possible anticipation in Brazilian facioscapulohumeral muscular dystrophy families.

A gene responsible for facioscapulohumeral muscular dystrophy (FSHD) has been localized at 4q35. Subsequently, it was found that probe p13E-11 detects a polymorphic EcoRI fragment, usually > 28 kb, in normal individuals, whereas in sporadic and familial FSHD cases, an EcoRI fragment, usually < 28 kb, was found. Although these findings have been amply confirmed, several aspects are as yet either controversial or unsolved. In the present investigation, 34 Brazilian FSHD families were studied at the clinical and the molecular level for the following purposes: to assess the frequency of new mutations and their effect on estimates of biological fitness, to characterize FSHD-associated EcoRI fragments detected with probe p13E-11 in familial--as compared with isolated--FSHD cases, and to assess whether anticipation occurs in multigenerational families. Results from our study suggest that new mutations are apparently frequent for FSHD and may account for at least one-third of the cases, that somatic mosaicism may not be rare, and that biological fitness appeared to be reduced in FSHD, ranging from 0.6 to 0.82 by different estimates, with no difference in sexes. Interestingly, the size of the new EcoRI fragment is apparently smaller in more severely affected isolated patients. Moreover, the age at onset of clinical signs, as well as the age at ascertainment, in patients from multigenerational families suggests that anticipation occurs for FSHD in the majority of the families.