Linkage disequilibrium at the Angelman syndrome gene UBE3A in autism families.

Autistic disorder is a neurodevelopmental disorder with a complex genetic etiology. Observations of maternal duplications affecting chromosome 15q11-q13 in patients with autism and evidence for linkage and linkage disequilibrium to markers in this region in chromosomally normal autism families indicate the existence of a susceptibility locus. We have screened the families of the Collaborative Linkage Study of Autism for several markers spanning a candidate region covering approximately 2 Mb and including the Angelman syndrome gene (UBE3A) and a cluster of gamma-aminobutyric acid (GABA(A)) receptor subunit genes (GABRB3, GABRA5, and GABRG3). We found significant evidence for linkage disequilibrium at marker D15S122, located at the 5' end of UBE3A. This is the first report, to our knowledge, of linkage disequilibrium at UBE3A in autism families. Characterization of null alleles detected at D15S822 in the course of genetic studies of this region showed a small (approximately 5-kb) genomic deletion, which was present at somewhat higher frequencies in autism families than in controls.     

A genomewide screen for autism: strong evidence for linkage to chromosomes 2q, 7q, and 16p

Autism is characterized by impairments in reciprocal communication and social interaction and by repetitive and stereotyped patterns of activities and interests. Evidence for a strong underlying genetic predisposition comes from twin and family studies, although susceptibility genes have not yet been identified. A whole-genome screen for linkage, using 83 sib pairs with autism, has been completed, and 119 markers have been genotyped in 13 candidate regions in a further 69 sib pairs. The addition of new families and markers provides further support for previous reports of linkages on chromosomes 7q and 16p. Two new regions of linkage have also been identified on chromosomes 2q and 17q. The most significant finding was a multipoint maximum LOD score (MLS) of 3.74 at marker D2S2188 on chromosome 2; this MLS increased to 4.80 when only sib pairs fulfilling strict diagnostic criteria were included. The susceptibility region on chromosome 7 was the next most significant, generating a multipoint MLS of 3.20 at marker D7S477. Chromosome 16 generated a multipoint MLS of 2.93 at D16S3102, whereas chromosome 17 generated a multipoint MLS of 2.34 at HTTINT2. With the addition of new families, there was no increased allele sharing at a number of other loci originally showing some evidence of linkage. These results support the continuing collection of multiplex sib-pair families to identify autism-susceptibility genes.     

The rates and patterns of deletions in the human factor IX gene.

Deletions are commonly observed in genes with either segments of highly homologous sequences or excessive gene length. However, in the factor IX gene and in most genes, deletions (of > or = 21 bp) are uncommon. We have analyzed DNA from 290 families with hemophilia B (203 independent mutations) and have found 12 deletions > 20 bp. Eleven of these are > 2 kb (range > 3-163 kb), and one is 1.1 kb. The junctions of the four deletions that are completely contained within the factor IX gene have been determined. A novel mutation occurred in patient HB128: the data suggest that a 26.8-kb deletion occurred between two segments of alternating purines and pyrimidines and that a 2.3-kb sense strand segment derived from the deleted region was inserted. For our sample of 203 independent mutations, we estimate the "baseline" rates of deletional mutation per base pair per generation as a function of size. The rate for large (> 2 kb) deletions is exceedingly low. For every mutational event in which a given base is at the junction of a large deletion, there are an estimated 58 microdeletions (< 20 bp) and 985 single-base substitutions at that base. Analysis of the nine reported deletion junctions in the factor IX gene literature reveals that (i) five are associated with inversions, orphan sequences, or sense strand insertions; (ii) four are simple deletions that display an excess of short direct repeats at their junctions; (iii) there is no dramatic clustering of junctions within the gene; and (iv) with the exception of alternating purines and pyrimidines, deletion junctions are not preferentially associated with repetitive DNA.     

Sequence structures of two developmentally regulated, alternative DNA deletion junctions in Tetrahymena thermophila.

Deletions of specific DNA sequences are known to occur in Tetrahymena thermophila as a developmentally regulated process. Deletions of a particular region (region M) were previously shown to be of two alternative sizes, 0.6 or 0.9 kilobases (kb) (C.F. Austerberry, C.D. Allis, and M.-C. Yao, Proc. Natl. Acad. Sci. USA 81: 7383-7387). In this study, the nucleotide sequences for both deletions were determined. These two deletions share the same right junction, but their left junctions are 0.3 kb apart. An 8-base-pair (bp) sequence is present at both junctions of the 0.6-kb deletion, but only 5 bp of this direct repeat are present at the left junction of the 0.9-kb deletion. Further comparison revealed a common 10-bp sequence near each of the two left junctions and a similar sequence in inverted orientation near the right junction. These sequences may play a role in the developmental regulation of the deletion process.     

Identification of a full-size hobo element and deletion-derivatives in Korean populations of Drosophila melanogaster.

We have isolated and characterized several members of the hobo transposable element family from Korean populations of Drosophila melanogaster. All of the Korean lines tested appeared to have 3.0 kb hobo elements and a high copy number of smaller derivatives of the element. To determine whether a 3.0 kb hobo element of these populations is consistent with the role of an autonomous hobo element, we cloned and sequenced this hobo element. Based on the result of the entire DNA sequence, a cloned 3.0 kb element called HKN96, it was found to be the same as a fully-functional 2959 bp HFL1-type sequence. Each small element appeared to have arisen from the HFL1 element by a different internal deletion. A specific 1.7 kb Kh hobo element, which is the most abundant in the Korean lines tested, seems to have originated from the HFL1 hobo element by an internal deletion of 1253 bp by the removal of nucleotides between positions 939 and 2191. The sequences of the Th1 and Th2 elements appeared to be identical to that of the HFL1 with the exception of internal deletions of 1442 bp and 1455 bp removing nucleotides 940-2381 and 923-2377, respectively. Based on the number of TPE repeats, all of the members of the hobo element family in Korean lines tested have three perfect S repeats. The widespread presence of identical copies of the Kh deletion derivative suggests that it might have a role in the regulation of hobo-induced hybrid dysgenesis.     

Microdeletion/microduplication of proximal 15q11.2 between BP1 and BP2: a susceptibility region for neurological dysfunction including developmental and language delay

The proximal long arm of chromosome 15 has segmental duplications located at breakpoints BP1?CBP5 that mediate the generation of NAHR-related microdeletions and microduplications. The classical Prader-Willi/Angelman syndrome deletion is flanked by either of the proximal BP1 or BP2 breakpoints and the distal BP3 breakpoint. The larger Type I deletions are flanked by BP1 and BP3 in both Prader-Willi and Angelman syndrome subjects. Those with this deletion are reported to have a more severe phenotype than individuals with either Type II deletions (BP2?CBP3) or uniparental disomy 15. The BP1?CBP2 region spans approximately 500?kb and contains four evolutionarily conserved genes that are not imprinted. Reports of mutations or disturbed expression of these genes appear to impact behavioral and neurological function in affected individuals. Recently, reports of deletions and duplications flanked by BP1 and BP2 suggest an association with speech and motor delays, behavioral problems, seizures, and autism. We present a large cohort of subjects with copy number alteration of BP1 to BP2 with common phenotypic features. These include autism, developmental delay, motor and language delays, and behavioral problems, which were present in both cytogenetic groups. Parental studies demonstrated phenotypically normal carriers in several instances, and mildly affected carriers in others, complicating phenotypic association and/or causality. Possible explanations for these results include reduced penetrance, altered gene dosage on a particular genetic background, or a susceptibility region as reported for other areas of the genome implicated in autism and behavior disturbances.     

Partial aldolase B gene deletions in hereditary fructose intolerance.

Hereditary fructose intolerance (HFI) is an autosomal recessive condition caused by a deficiency of aldolase B. We have recently shown that three point mutations in this gene account for approximately 85% of HFI alleles in Europe and the United States and are thus of diagnostic importance. In this paper we define three new lesions in the aldolase B gene: two are large deletions, one of 1.65 kb and one of 1.4 kb; the third is a small deletion of 4 bp. We have determined the breakpoints of these deletions and have demonstrated that the presence of such lesions may complicate the genotyping of individuals for diagnosis of HFI.     

Linkage disequilibrium analysis in Machado-Joseph disease patients of different ethnic origins

Machado-Joseph disease (MJD) is an autosomal dominant spinocerebellar degeneration originally described in families of Portuguese-Azorean ancestry. The hypothesis that its present world distribution could result from the spread of an original founder mutation has been raised. To test this possibility we have conducted a linkage disequilibrium study of markers segregating with the MJD1 locus in a total of 64 unrelated families of different geographical origins. Significant association was detected between the MJD1 locus and marker alleles at loci D14S280, D14S1050 and D14S81. All affected individuals, except one Chinese family, had allele 3 (237 bp) at D14S280. This finding is consistent with a founder effect in our MJD population. However, distinct haplotypes were observed in patients originating from the two Azorean islands showing the highest disease prevalence; therefore, the possible existence of more than one founder mutation can not be excluded with the markers currently available. Received: 27 February 1996 / Revised: 4 June 1996     

Recombinations between Alu repeat sequences that result in partial deletions within the C1 inhibitor gene

Genomic DNA sequence analysis was used to define the extent of deletions within the C1 inhibitor gene in two families with type I hereditary angioneurotic edema. Southern blot analysis initially indicated the presence of the partial deletions. One deletion was approximately 2 kb and included exon VII, whereas the other was approximately 8.5 kb and included exons IV–VI. Genomic libraries from an affected member of each family were constructed and clones containing the deletions were analyzed. Sequence analysis of the deletion joints of the mutants and corresponding regions of the normal gene in the two families demonstrated that both deletion joints resulted from recombination of two Alu repetitive DNA elements. Alu repeat sequences from introns VI and VII combined to make a novel Alu in family A, and Alu sequences in introns III and VI were spliced to make a new Alu in family B. The splice sites in the Alu sequences of both mutants were located in the left arm of the Alu element, and both recombination joints overlapped one of the RNA polymerase III promoter sequences. Because the involved Alu sequences, in both instances, were oriented in the same direction, unequal crossingover is the most likely mechanism to account for these mutations.     

Molecular analysis of 24 Alagille syndrome families identifies a single submicroscopic deletion and further localizes the Alagille region within 20p12.

Alagille syndrome (AGS) is a clinically defined disorder characterized by cholestatic liver disease with bile duct paucity, peculiar facies, structural heart defects, vertebral anomalies, and ocular abnormalities. Multiple patients with various cytogenetic abnormalities involving 20p12 have been identified, allowing the assignment of AGS to this region. The presence of interstitial deletions of varying size led to the hypothesis that AGS is a contiguous gene deletion syndrome. This molecular analysis of cytogenetically normal AGS patients was performed in order to test this hypothesis and to refine the localization of the known AGS region. Investigation of inheritance of simple tandem repeat polymorphism alleles in 67 members of 24 cytogenetically normal Alagille families led to the identification of a single submicroscopic deletion. The deletion included loci D20S61, D20S41, D20S186, and D20S188 and presumably intervening uninformative loci D20S189 and D20S27. The six deleted loci are contained in a single YAC of 1.9 Mb. The additional finding of multiple unrelated probands who are heterozygous at each locus demonstrates that microdeletions at known loci within the AGS region are rare in cytogenetically normal patients with this disorder. This suggests that the majority of cases of AGS may be the result of a single gene defect rather than a contiguous gene deletion syndrome.     

Linkage analysis in familial Angelman syndrome.

Familial Angelman syndrome (AS) can result from mutations in chromosome 15q11q13 that, when transmitted from father to child, result in no phenotypic abnormality but, when transmitted from mother to child, cause AS. These mutations therefore behave neither as dominant nor as recessive mutations but, rather, show an imprinted mode of inheritance. We have analyzed two sibling pairs with AS and a larger family with four AS offspring of three sisters with several recently described microsatellite polymorphisms in the AS region. AS siblings inherited the same maternal alleles at the GABRB3 and GABRA5 loci, and the unaffected siblings of AS individuals inherited the other maternal alleles at these loci. In one of the AS sibling pairs, analysis of a recombination event indicates that the mutation responsible for AS is distal to locus D15S63. This result is consistent with a previously described imprinted submicroscopic deletion causing AS, a deletion that includes loci D15S10, D15S113, and GABRB3, all distal to D15S63. The analysis of the larger AS family provides the first clear demonstration of a new mutation in nondeletion AS. Analysis of linkage of AS to GABRB3 in these three families, on the assumption of imprinted inheritance (i.e., penetrance of an AS mutation is 1 if transmitted maternally and is 0 if transmitted paternally), indicates a maximum lod score of 3.52 at theta = 0.     

Isolation of Caenorhabditis elegans gene knockouts by PCR screening of chemically mutagenized libraries

This protocol details methodologies to generate Caenorhabditis elegans deletion mutants by chemical mutagenesis and to detect them by PCR screening. Approximately, 600,000 worms are grown synchronously, mutagenized with ethyl methane sulfonate, divided in groups of 500 and allowed to self-fertilize for two generations. DNA is prepared from a fraction of each worm population, pooled into a 96-well plate, and screened by PCR with primers positioned 2.5-3.5 kb apart. Cultures containing deletion mutants are subdivided in small worm populations and tested again by PCR to identify positives. Single animals are then cloned from positive cultures, allowed to self-fertilize and identified by PCR genotyping. This method, which takes about a month, gives approximately a 50% chance of finding a deletion of interest larger than 500-600 bp. If a deletion cannot be found, the library can be pooled at lower complexity and screened for smaller deletions using an alternative PCR-based method.     

Mutations that uncouple the beta-adrenergic receptor from Gs and increase agonist affinity

The deletion of residues 239-272 from the hamster beta-adrenergic receptor resulted in a loss of the ability of the receptor, expressed in mouse L cells, to stimulate adenylate cyclase (Dixon, R. A. F., Sigal, I. S., Rands, E., Register, R. B., Candelore, M. R., Blake, A. D., and Strader, C. D. (1987) Nature 326, 73-77). This mutant receptor (D(239-272)beta AR) bound the agonist isoproterenol with a single class of binding sites, in contrast to the wild-type beta-adrenergic receptor, which exhibited two classes of agonist affinity sites. We now report that the affinity of D(239-272)beta AR for isoproterenol is relatively insensitive to detergent solubilization or to treatment with either GTP or NaF, indicating the absence of a receptor-Gs interaction. Whereas deletions within the region of amino acids 229-258 did not reduce the ability of the receptor to couple to Gs or to stimulate adenylate cyclase, the deletion of either of the regions 222-229 or 258-270 resulted in receptors which were unable to couple to Gs. The affinities of D(222-229)beta AR, D(239-272)beta AR, and D(258-270)beta AR toward isoproterenol were greater than that observed for the low affinity, uncoupled form of the wild-type receptor. These results suggest a role for the regions of the beta-adrenergic receptor encompassing amino acids 222-229 and 258-270, which are predicted to form amphiphilic helices, in the agonist-promoted activation of Gs.     

Familial Chordoma, a Tumor of Notochordal Remnants, Is Linked to Chromosome 7q33

Chordoma is a rare tumor originating from notochordal remnants that is usually diagnosed during midlife. We performed a genomewide analysis for linkage in a family with 10 individuals affected by chordoma. The maximum two-point LOD score based on only the affected individuals was 2.21, at recombination fraction 0, at marker D7S2195 on chromosome 7q. Combined analysis of additional members of this family (11 affected individuals) and of two unrelated families (one with 2 affected individuals and the other with 3 affected individuals), with 20 markers on 7q, showed a maximum two-point LOD score of 4.05 at marker D7S500. Multipoint analysis based on only the affected individuals gave a maximum LOD score of 4.78, with an approximate 2-LOD support interval from marker D7S512 to marker D7S684. Haplotype analysis of the three families showed a minimal disease-gene region from D7S512 to D7S684, a distance of 11.1 cM and approximately 7.1 Mb. No loss of heterozygosity was found at markers D7S1804, D7S1824, and D7S2195 in four tumor samples from affected family members. These results map a locus for familial chordoma to 7q33. Further analysis of this region, to identify this gene, is ongoing.     

Molecular definition of the chromosome 7 deletion in Williams syndrome and parent-of-origin effects on growth.

Williams syndrome (WS) is a developmental disorder with variable phenotypic expression associated, in most cases, with a hemizygous deletion of part of chromosomal band 7q11.23 that includes the elastin gene (ELN). We have investigated the frequency and size of the deletions, determined the parental origin, and correlated the molecular results with the clinical findings in 65 WS patients. Hemizygosity at the ELN locus was established by typing of two intragenic polymorphisms, quantitative Southern analysis, and/or FISH. Polymorphic markers covering the deletion and flanking regions were ordered by a combination of genetic and physical mapping. Genotyping of WS patients and available parents for 13 polymorphisms revealed that of 65 clinically defined WS patients, 61 (94%) had a deletion of the ELN locus and were also hemizygous (or noninformative) at loci D7S489B, D7S2476, D7S613, D7S2472, and D7S1870. None of the four patients without ELN deletion was hemizygous at any of the polymorphic loci studied. All patients were heterozygous (or noninformative) for centromeric (D7S1816, D7S1483, and D7S653) and telomeric (D7S489A, D7S675, and D7S669) flanking loci. The genetic distance between the most-centromeric deleted locus, D7S489B, and the most-telomeric one, D7S1870, is 2 cM. The breakpoints cluster at approximately 1 cM to either side of ELN. In 39 families informative for parental origin, all deletions were de novo, and 18 were paternally and 21 maternally derived. Comparison of clinical data, collected in a standardized quantifiable format, revealed significantly more severe growth retardation and microcephaly in the maternal deletion group. An imprinted locus, silent on the paternal chromosome and contributing to statural growth, may be affected by the deletion.     

Y-chromosome specific alleles and haplotypes in European and Asian populations: Linkage disequilibrium and geographic diversity

Variation on the Y chromosome may permit our understanding the evolution of the human paternal lineage and male gene flow. This study reports upon the distribution and non random association of alleles at four Y-chromosome specific loci in four populations, three Caucasoid (Italian, Greek and Slav) and one Asian. The markers include insertion/deletion (p12f), point mutation (92R7 and pYαI), and repeat sequence (p21A1) polymorphisms. Our data confirm that the p12f/TaqI 8 kb allele is a Caucasoid marker and that Asians are monomorphic at three of the loci (p12f, 92R7, and pYαI). The alleles at 92R7 and pYαI were found to be in complete disequilibrium in Europeans. Y-haplotype diversity was highly significant between Asians and all three European groups (P < 0.001), but the Greeks and Italians were also significantly different with respect to some alleles and haplotypes (P < 0.02). We find strong evidence that the p12f/TaqI 8 kb allele may have arisen only once, as a deletion event, and, additionally, that the present-day frequency distribution of Y chromosomes carrying the p12f/8 kb allele suggests that it may have been spread by colonising sea-faring peoples from the Near East, possibly the Phoenicians, rather than by expansion of Neolithic farmers into continental Europe. The p12f deletion is the key marker of a unique Y chromosome, found only in Caucasians to date, labelled ‘Mediterranean’ and this further increases the level of Y-chromosome diversity seen among Caucasoids when compared to the other major population groups. Am J Phys Anthropol 104:167–176, 1997. © 1997 Wiley-Liss, Inc.     

Characterization of Genomic Deletion Efficiency Mediated by Clustered Regularly Interspaced Palindromic Repeats (CRISPR)/Cas9 Nuclease System in Mammalian Cells

The clustered regularly interspaced palindromic repeats (CRISPR)/CRISPR-associated (Cas) 9 nuclease system has provided a powerful tool for genome engineering. Double strand breaks may trigger nonhomologous end joining repair, leading to frameshift mutations, or homology-directed repair using an extrachromosomal template. Alternatively, genomic deletions may be produced by a pair of double strand breaks. The efficiency of CRISPR/Cas9-mediated genomic deletions has not been systematically explored. Here, we present a methodology for the production of deletions in mammalian cells, ranging from 1.3 kb to greater than 1 Mb. We observed a high frequency of intended genomic deletions. Nondeleted alleles are nonetheless often edited with inversions or small insertion/deletions produced at CRISPR recognition sites. Deleted alleles also typically include small insertion/deletions at predicted deletion junctions. We retrieved cells with biallelic deletion at a frequency exceeding that of probabilistic expectation. We demonstrate an inverse relationship between deletion frequency and deletion size. This work suggests that CRISPR/Cas9 is a robust system to produce a spectrum of genomic deletions to allow investigation of genes and genetic elements.