Uncommon deletions of the Smith-Magenis syndrome region can be recurrent when alternate low-copy repeats act as homologous recombination substrates

Several homologous recombination "hotspots," or sites of positional preference for strand exchanges, associated with recurrent deletions and duplications have been reported within large low-copy repeats (LCRs). Recently, such a hotspot was identified in patients with the Smith-Magenis syndrome (SMS) common deletion of approximately 4 Mb or a reciprocal duplication within the KER gene cluster of the SMS-REP LCRs, in which 50% of analyzed strand exchanges resulting in deletion and 23% of those resulting in duplication occurred. Here, we report an additional recombination hotspot within LCR17pA and LCR17pD, which serve as alternative substrates for nonallelic homologous recombination that results in large (approximately 5 Mb) deletions of 17p11.2, which include the SMS region. Using polymerase-chain-reaction mapping of somatic cell hybrid lines, we refined the breakpoints of six deletions within these LCRs. Sequence analysis of the recombinant junctions revealed that all six strand exchanges occurred within a 524-bp interval, and four of them occurred within an AluSq/x element. This interval represents only 0.5% of the 124-kb stretch of 98.6% sequence identity between LCR17pA and LCR17pD. A search for potentially stimulating sequence motifs revealed short AT-rich segments flanking the recombination hotspot. Our findings indicate that alternative LCRs can mediate rearrangements, resulting in haploinsufficiency of the SMS critical region, and reimplicate homologous recombination as a major mechanism for genomic disorders.     

Molecular characterization of a 130-kb terminal microdeletion at 22q in a child with mild mental retardation.

We have analyzed a recently described 22q13.3 microdeletion in a child with some overlapping features of the cytologically visible 22q13.3 deletion syndrome. Patient NT, who shows mild mental retardation and delay of expressive speech, was previously found to have a paternal microdeletion in the subtelomeric region of 22q. In order to characterize this abnormality further, we have constructed a cosmid/P1 contig covering the terminal 150 kb of 22q, which encompasses the 130-kb microdeletion. The microdeletion breakpoint is within the VNTR locus D22S163. The cloning of the breakpoint sequence revealed that the broken chromosome end was healed by the addition of telomeric repeats, indicating that the microdeletion is terminal. This is the first cloned terminal deletion breakpoint on a human chromosome other than 16p. The cosmid/P1 contig was mapped by pulsed-field gel electrophoresis analysis to within 120 kb of the arylsulfatase A gene, which places the contig in relation to genetic and physical maps of the chromosome. The acrosin gene maps within the microdeletion, approximately 70 kb from the telomere. With the distal end of chromosome 22q cloned, it is now possible to isolate genes that may be involved in the overlapping phenotype of this microdeletion and 22q13.3 deletion syndrome.     

The donor chromosome breakpoint for a jumping translocation is associated with large low-copy repeats in 21q21.3

Jumping translocations (JTs) are very rare chromosome aberrations, usually identified in tumors. We report a constitutional JT between donor chromosome 21q21.3-->qter and recipients 13qter and 18qter, resulting in an approximately 15.5-Mb proximal deletion 21q in a girl with mild developmental delay and minor dysmorphic features. Using fluorescence in situ hybridization (FISH) studies, we identified an approximately 550-kb complex inter- and intra-chromosomal low-copy repeat (LCR) adjacent to the 21q21.3 translocation breakpoint. On the recipient chromosomes 13qter and 18qter, the telomeric sequences TTAGGG were retained. Genotyping revealed that the deletion was of maternal origin. We propose that genome architecture involving LCRs may be a major mechanism responsible for the origin of jumping translocations.     

Preferential paternal origin of microdeletions caused by prezygotic chromosome or chromatid rearrangements in Sotos syndrome

Sotos syndrome (SoS) is characterized by pre- and postnatal overgrowth with advanced bone age; a dysmorphic face with macrocephaly and pointed chin; large hands and feet; mental retardation; and possible susceptibility to tumors. It has been shown that the major cause of SoS is haploinsufficiency of the NSD1 gene at 5q35, because the majority of patients had either a common microdeletion including NSD1 or a truncated type of point mutation in NSD1. In the present study, we traced the parental origin of the microdeletions in 26 patients with SoS by the use of 16 microsatellite markers at or flanking the commonly deleted region. Deletions in 18 of the 20 informative cases occurred in the paternally derived chromosome 5, whereas those in the maternally derived chromosome were found in only two cases. Haplotyping analysis of the marker loci revealed that the paternal deletion in five of seven informative cases and the maternal deletion in one case arose through an intrachromosomal rearrangement, and two other cases of the paternal deletion involved an interchromosomal event, suggesting that the common microdeletion observed in SoS did not occur through a uniform mechanism but preferentially arose prezygotically.     

Reciprocal crossovers and a positional preference for strand exchange in recombination events resulting in deletion or duplication of chromosome 17p11.2

Smith-Magenis syndrome (SMS) is caused by an approximately 4-Mb heterozygous interstitial deletion on chromosome 17p11.2 in approximately 80%-90% of affected patients. Three large ( approximately 200 kb), complex, and highly homologous ( approximately 98%) low-copy repeats (LCRs) are located inside or flanking the SMS common deletion. These repeats, also known as "SMS-REPs," are termed "distal," "middle," and "proximal." The directly oriented distal and proximal copies act as substrates for nonallelic homologous recombination resulting in both the deletion associated with SMS and the reciprocal duplication: dup(17)(p11.2p11.2). Using restriction enzyme cis-morphism analyses and direct sequencing, we mapped the regions of strand exchange in 16 somatic-cell hybrids that harbor only the recombinant SMS-REP. Our studies showed that the sites of crossovers were distributed throughout the region of homology between the distal and proximal SMS-REPs. However, despite approximately 170 kb of high homology, 50% of the recombinant junctions occurred in a 12.0-kb region within the KER gene clusters. DNA sequencing of this hotspot (positional preference for strand exchange) in seven recombinant SMS-REPs narrowed the crossovers to an approximately 8-kb interval. Four of them occurred in a 1,655-bp region rich in polymorphic nucleotides that could potentially reflect frequent gene conversion. For further evaluation of the strand exchange frequency in patients with SMS, novel junction fragments from the recombinant SMS-REPs were identified. As predicted by the reciprocal-recombination model, junction fragments were also identified from this hotspot region in patients with dup(17)(p11.2p11.2), documenting reciprocity of the positional preference for strand exchange. Several potential cis-acting recombination-promoting sequences were identified within the hotspot. It is interesting that we found 2.1-kb AT-rich inverted repeats flanking the proximal and middle KER gene clusters but not the distal one. The role of any or all of these in stimulating double-strand breaks around this positional recombination hotspot remains to be explored.     

Characterization of a novel microdeletion polymorphism on BTA5 in cattle

We present a detailed breakpoint mapping and population frequency analysis of a 214-kb microdeletion that removes multiple olfactory receptor genes. Using progressive rounds of PCR assays, we mapped the upstream and downstream breakpoints of this microdeletion event to approximately 1 and 12 kb genomic regions, respectively. We developed PCR-based genotyping assays, characterized a dairy cattle panel of 96 samples and found that the frequency of the deletion allele was over 51%. Our results indicated that this microdeletion is an ancient event occurring in one of the earlier founders, and that it has been stably inherited across generations in the North American dairy cattle population.     

Mutational mechanisms of Williams-Beuren syndrome deletions

Williams-Beuren syndrome (WBS) is a segmental aneusomy syndrome that results from a heterozygous deletion of contiguous genes at 7q11.23. Three large region-specific low-copy repeat elements (LCRs), composed of different blocks (A, B, and C), flank the WBS deletion interval and are thought to predispose to misalignment and unequal crossing-over, causing the deletions. In this study, we have determined the exact deletion size and LCR copy number in 74 patients with WBS, as well as precisely defined deletion breakpoints in 30 of them, using LCR-specific nucleotide differences. Most patients (95%) exhibit a 1.55-Mb deletion caused by recombination between centromeric and medial block B copies, which share approximately 99.6% sequence identity along 105-143 kb. In these cases, deletion breakpoints were mapped at several sites within the recombinant block B, with a cluster (>27%) occurring at a 12 kb region within the GTF2I/GTF2IP1 gene. Almost one-third (28%) of the transmitting progenitors were found to be heterozygous for an inversion between centromeric and telomeric LCRs. All deletion breakpoints in the patients with the inversion occurred in the distal 38-kb block B region only present in the telomeric and medial copies. Finally, only four patients (5%) displayed a larger deletion ( approximately 1.84 Mb) caused by recombination between centromeric and medial block A copies. We propose models for the specific pairing and precise aberrant recombination leading to each of the different germline rearrangements that occur in this region, including inversions and deletions associated with WBS. Chromosomal instability at 7q11.23 is directly related to the genomic structure of the region.     

Fine mapping of the constitutional translocation t(11;22)(q23;q11)

Translocation t(11;22)(q23;q11) is the most common constitutional reciprocal translocation in man. Balanced carriers are phenotypically normal, except for decreased fertility, an increased spontaneous abortion rate and a possible predisposition to breast cancer in some families. Here, we report the high resolution mapping of the t(11;22)(q23;q11) breakpoint. We have localised the breakpoint, by using fluorescence in situ hybidisation (FISH) walking, to a region between D11S1340 and WI-8564 on chromosome 11, and D22S134 and D22S264 on chromosome 22. We report the isolation of a bacterial artificial chromosome (BAC) clone spanning the breakpoint in 11q23. We have narrowed down the breakpoint to an 80-kb sequenced region on chromosome 11 and FISH analysis has revealed a variation of the breakpoint position between patients. In 22q11, we have sequenced two BACs (BAC2280L11 and BAC41C4) apparently mapping to the region; these contain low copy repeats (LCRs). Southern blot analysis with probes from BAC2280L11 has revealed different patterns between normal controls and translocation carriers, indicating that sequences similar/identical to these probes flank the translocation breakpoint. The occurrence of LCRs has previously been associated with genomic instability and "unclonable" regions. Hence, the presence of such repeats renders standard translocation breakpoint cloning techniques ineffective. Thus, we have used high resolution fiber-FISH to study this region in normal and translocation cases by using probes from 22q11, LCRs and 11q23. We demonstrate that the LCR containing the gap in 22q11 is probably substantially larger than the previous estimates of 100 kb. Using fiber-FISH, we have localised the breakpoint in 22q11 to approximately 20-40 kb from the centromeric border of the LCR (i.e. the telomeric end of AC006547) and have confirmed the breakpoint position on 11q23.     

Molecular basis of Sotos syndrome

This paper describes the isolation of a novel human gene, NSD1, from the 5q35 breakpoint of t(5;8)(q35; q24.1) in a patient with Sotos syndrome, and NSD1 mutation analysis. Of 112 (95 Japanese and 17 non-Japanese) patients analyzed, 16 (14%) had a heterozygous NSD1 point mutation (10 protein truncation types and six missense types) and 50 (45%) a approximately 0.7-Mb microdeletion involving NSD1. The results indicated that haploinsufficiency of NSD1 is the major cause of Sotos syndrome, and NSD1 plays a role in growth and brain development in humans. Detailed clinical examinations provided a genotype-phenotype correlation in Sotos syndrome, i.e. in patients with deletions, overgrowth is less obvious and mental retardation is more severe than in those with point mutations, and major anomalies were exclusively seen in the former. The results also indicated that Sotos syndrome due to a deletion falls into a contiguous gene syndrome, while Sotos syndrome due to an NSD1 point mutation is a single gene defect, occasionally with an autosomal dominant mode of inheritance. The genomic structure around the deleted and flanking regions revealed the presence of two sets of low copy repeats through which the microdeletion in Sotos syndrome is mediated.     

Characterization of the NPHP1 locus: mutational mechanism involved in deletions in familial juvenile nephronophthisis

Familial juvenile nephronophthisis is an autosomal recessive, genetically heterogeneous kidney disorder representing the most frequent inherited cause of chronic renal failure in children. A gene, NPHP1, responsible for approximately 85% of the purely renal form of nephronophthisis, has been mapped to 2q13 and characterized. The major NPHP1 gene defect is a large homozygous deletion found in approximately 80% of the patients. In this study, by large-scale genomic sequencing and pulsed-field gel electrophoresis analysis, we characterized the complex organization of the NPHP1 locus and determined the mutational mechanism that results in the large deletion observed in most patients. We showed that the deletion is 290 kb in size and that NPHP1 is flanked by two large inverted repeats of approximately 330 kb. In addition, a second sequence of 45 kb located adjacent to the proximal 330-kb repeat was shown to be directly repeated 250 kb away within the distal 330-kb repeat deleting the sequence tag site (STS) 804H10R present in the proximal copy. The patients' deletion breakpoints appear to be located within the 45-kb repeat, suggesting an unequal recombination between the two homologous copies of this smaller repeat. Moreover, we demonstrated a nonpathologic rearrangement involving the two 330-kb inverted repeats found in 11 patients and, in the homozygous state, in 2 (1.3%) control individuals. This could be explained by interchromosomal mispairing of the 330-kb inverted repeat, followed by double recombination or by a prior intrachromosomal mispairing of these repeats, leading to an inversion of the NPHP1 region, followed by an interchromosomal unequal crossover event. This complex rearrangement, as well as the common deletion found in most patients, illustrates the high level of rearrangements occurring in the centromeric region of chromosome 2.     

Genome architecture catalyzes nonrecurrent chromosomal rearrangements

To investigate the potential involvement of genome architecture in nonrecurrent chromosome rearrangements, we analyzed the breakpoints of eight translocations and 18 unusual-sized deletions involving human proximal 17p. Surprisingly, we found that many deletion breakpoints occurred in low-copy repeats (LCRs); 13 were associated with novel large LCR17p structures, and 2 mapped within an LCR sequence (middle SMS-REP) within the Smith-Magenis syndrome (SMS) common deletion. Three translocation breakpoints involving 17p11 were found to be located within the centromeric alpha-satellite sequence D17Z1, three within a pericentromeric segment, and one at the distal SMS-REP. Remarkably, our analysis reveals that LCRs constitute >23% of the analyzed genome sequence in proximal 17p--an experimental observation two- to fourfold higher than predictions based on virtual analysis of the genome. Our data demonstrate that higher-order genomic architecture involving LCRs plays a significant role not only in recurrent chromosome rearrangements but also in translocations and unusual-sized deletions involving 17p.     

22q11.2 distal deletion: a recurrent genomic disorder distinct from DiGeorge syndrome and velocardiofacial syndrome

Microdeletions within chromosome 22q11.2 cause a variable phenotype, including DiGeorge syndrome (DGS) and velocardiofacial syndrome (VCFS). About 97% of patients with DGS/VCFS have either a common recurrent ~3 Mb deletion or a smaller, less common, ~1.5 Mb nested deletion. Both deletions apparently occur as a result of homologous recombination between nonallelic flanking low-copy repeat (LCR) sequences located in 22q11.2. Interestingly, although eight different LCRs are located in proximal 22q, only a few cases of atypical deletions utilizing alternative LCRs have been described. Using array-based comparative genomic hybridization (CGH) analysis, we have detected six unrelated cases of deletions that are within 22q11.2 and are located distal to the ~3 Mb common deletion region. Further analyses revealed that the rearrangements had clustered breakpoints and either a ~1.4 Mb or ~2.1 Mb recurrent deletion flanked proximally by LCR22-4 and distally by either LCR22-5 or LCR22-6, respectively. Parental fluorescence in situ hybridization (FISH) analyses revealed that none of the available parents (11 out of 12 were available) had the deletion, indicating de novo events. All patients presented with characteristic facial dysmorphic features. A history of prematurity, prenatal and postnatal growth delay, developmental delay, and mild skeletal abnormalities was prevalent among the patients. Two patients were found to have a cardiovascular malformation, one had truncus arteriosus, and another had a bicuspid aortic valve. A single patient had a cleft palate. We conclude that distal deletions of chromosome 22q11.2 between LCR22-4 and LCR22-6, although they share some characteristic features with DGS/VCFS, represent a novel genomic disorder distinct genomically and clinically from the well-known DGS/VCF deletion syndromes.     

Segmental duplication implicated in the genesis of inversion 2Rj of Anopheles gambiae

The malaria vector Anopheles gambiae maintains high levels of inversion polymorphism that facilitate its exploitation of diverse ecological settings across tropical Africa. Molecular characterization of inversion breakpoints is a first step toward understanding the processes that generate and maintain inversions. Here we focused on inversion 2Rj because of its association with the assortatively mating Bamako chromosomal form of An. gambiae, whose distinctive breeding sites are rock pools beside the Niger River in Mali and Guinea. Sequence and computational analysis of 2Rj revealed the same 14.6 kb insertion between both breakpoints, which occurred near but not within predicted genes. Each insertion consists of 5.3 kb terminal inverted repeat arms separated by a 4 kb spacer. The insertions lack coding capacity, and are comprised of degraded remnants of repetitive sequences including class I and II transposable elements. Because of their large size and patchwork composition, and as no other instances of these insertions were identified in the An. gambiae genome, they do not appear to be transposable elements. The 14.6 kb modules inserted at both 2Rj breakpoint junctions represent low copy repeats (LCRs, also called segmental duplications) that are strongly implicated in the recent (approximately 0.4N(e) generations) origin of 2Rj. The LCRs contribute to further genome instability, as demonstrated by an imprecise excision event at the proximal breakpoint of 2Rj in field isolates.     

An Alu-mediated large deletion of the FUT2 gene in individuals with the ABO-Bombay phenotype

Recently, we have found an allelic deletion of the secretor alpha(1,2)fucosyltransferase (FUT2) gene in individuals with the classical Bombay phenotype of the ABO system. The FUT2 gene consists of two exons separated by an intron that spans approximately 7 kb. The first exon is noncoding, whereas exon 2 contains the complete coding sequence. Since the 5' breakpoint of the deletion has previously been mapped to the single intron of FUT2, we have cloned the junction region of the deletion in a Bombay individual by cassette-mediated polymerase chain reaction. In addition, the region from the 3' untranslated region of FUT2 to the 3' breakpoint sequence has been amplified from a control individual. DNA sequence analysis of this region indicates that the 5' breakpoint is within a free left Alu monomer (FLAM-C) sequence that lies 1.3 kb downstream of exon 1, and that the 3' breakpoint is within a complete Alu element (AluSx) that is positioned 1.5 kb downstream of exon 2. The size of the deletion is estimated to be about 10 kb. There is a 25-bp sequence identity between the reference DNA sequences surrounding the 5' and 3' breakpoints. This demonstrates that an Alu-mediated large gene deletion generated by unequal crossover is responsible for secretor alpha(1,2)fucosyltransferase deficiency in Indian Bombay individuals.     

Computational analysis and refinement of sequence structure on chromosome 22q11.2 region: application to the development of quantitative real-time PCR assay for clinical diagnosis

The low-copy repeat (LCR) is a new class of repetitive DNA element and has been implicated in many human disorders, including DiGeorge/velocardiofacial syndrome (DGS/VCFS). It is now recognized that nonallelic homologous recombination (NAHR) through LCRs flanking the chromosome 22q11.2 region leads to genome rearrangements and results in the DGS/VCFS. To refine the structure and content of chromosome 22q11.2 LCRs, we applied computational analysis to dissect region-specific LCRs using publicly available sequences. Nine distinct duplicons between 1.6 and 65 kb long and sharing >95% sequence identity were identified. The presence of these sequence motifs supports the NAHR mechanism. Further sequence analysis suggested that the previously defined 3-Mb deletion may actually comprise two deletion intervals of similar size close to each other and thus indistinguishable when using fluorescence in situ hybridization (FISH) analysis. The differentially deleted regions contain several hypothetical proteins and UniGene clusters and may partially explain the clinical heterogeneity observed in DGS/VCFS patients with the 3-Mb common deletion. To implement further sequence information in molecular medicine, we designed a real-time quantitative PCR assay and validated the method in 122 patients with suspected DGS/VCFS. The assay detected 28 patients with chromosome 22q11.2 deletion later confirmed using FISH. Our results indicated that the developed assay is reliable as well as time and cost effective for clinical diagnosis of chromosome 22q11.2 deletion. They also suggest that this methodology can be applied to develop a molecular approach for clinical detection and diagnosis of other genomic disorders.     

Microduplication 22q11.2, an emerging syndrome: clinical, cytogenetic, and molecular analysis of thirteen patients

Chromosome 22, particularly band 22q11.2, is predisposed to rearrangements due to misalignments of low-copy repeats (LCRs). DiGeorge/velocardiofacial syndrome (DG/VCFS) is a common disorder resulting from microdeletion within the same band. Although both deletion and duplication are expected to occur in equal proportions as reciprocal events caused by LCR-mediated rearrangements, very few microduplications have been identified. We have identified 13 cases of microduplication 22q11.2, primarily by interphase fluorescence in situ hybridization (FISH). The size of the duplications, determined by FISH probes from bacterial artificial chromosomes and P(1) artificial chromosomes, range from 3-4 Mb to 6 Mb, and the exchange points seem to involve an LCR. Molecular analysis based on 15 short tandem repeats confirmed the size of the duplications and indicated that at least 1 of 15 loci has three alleles present. The patients' phenotypes ranged from mild to severe, sharing a tendency for velopharyngeal insufficiency with DG/VCFS but having other distinctive characteristics, as well. Although the present series of patients was ascertained because of some overlapping features with DG/VCF syndromes, the microduplication of 22q11.2 appears to be a new syndrome.     

Analysis of genes for 5S rRNA from the cricket, Acheta domesticus: two classes of repeating units

To examine the modulation of 5S rRNA gene activity during development in the cricket, Acheta domesticus, 5S X DNA was isolated from a lambda Charon 4 genomic library and characterized. Southern blot analysis of cloned A. domesticus genomic DNA revealed that restriction fragments of 3.0 and 2.1 kb represent two size classes of 5S X DNA repeating units; over 90% of the repeats measure 3.0 kb. Restriction analysis of two 5S X DNA clones suggests that the 2.1-kb repeats are not randomly interspersed within clusters of the larger 3.0-kb repeating units. Heteroduplex and restriction mapping of several clones indicate that the spacers of both repeating units account for their unusual length. The major difference between the two classes of repeats may lie in 0.9-kb spacer sequences to the 3.0-kb repeats.     

3q29 microdeletion syndrome: clinical and molecular characterization of a new syndrome

We report the identification of six patients with 3q29 microdeletion syndrome. The clinical phenotype is variable despite an almost identical deletion size. The phenotype includes mild-to-moderate mental retardation, with only slightly dysmorphic facial features that are similar in most patients: a long and narrow face, short philtrum, and high nasal bridge. Autism, gait ataxia, chest-wall deformity, and long and tapering fingers were noted in at least two of six patients. Additional features--including microcephaly, cleft lip and palate, horseshoe kidney and hypospadias, ligamentous laxity, recurrent middle ear infections, and abnormal pigmentation--were observed, but each feature was only found once, in a single patient. The microdeletion is approximately 1.5 Mb in length, with molecular boundaries mapping within the same or adjacent bacterial artificial chromosome (BAC) clones at either end of the deletion in all patients. The deletion encompasses 22 genes, including PAK2 and DLG1, which are autosomal homologues of two known X-linked mental retardation genes, PAK3 and DLG3. The presence of two nearly identical low-copy repeat sequences in BAC clones on each side of the deletion breakpoint suggests that nonallelic homologous recombination is the likely mechanism of disease causation in this syndrome.