The Alu-rich genomic architecture of SPAST predisposes to diverse and functionally distinct disease-associated CNV alleles.

Intragenic copy-number variants (CNVs) contribute to the allelic spectrum of both Mendelian and complex disorders. Although pathogenic deletions and duplications in SPAST (mutations in which cause autosomal-dominant spastic paraplegia 4 [SPG4]) have been described, their origins and molecular consequences remain obscure. We mapped breakpoint junctions of 54 SPAST CNVs at nucleotide resolution. Diverse combinations of exons are deleted or duplicated, highlighting the importance of particular exons for spastin function. Of the 54 CNVs, 38 (70%) appear to be mediated by an Alu-based mechanism, suggesting that the Alu-rich genomic architecture of SPAST renders this locus susceptible to various genome rearrangements. Analysis of breakpoint Alus further informs a model of Alu-mediated CNV formation characterized by small CNV size and potential involvement of mechanisms other than homologous recombination. Twelve deletions (22%) overlap part of SPAST and a portion of a nearby, directly oriented gene, predicting novel chimeric genes in these subjects' genomes. cDNA from a subject with a SPAST final exon deletion contained multiple SPAST:SLC30A6 fusion transcripts, indicating that SPAST CNVs can have transcriptional effects beyond the gene itself. SLC30A6 has been implicated in Alzheimer disease, so these fusion gene data could explain a report of spastic paraplegia and dementia cosegregating in a family with deletion of the final exon of SPAST. Our findings provide evidence that the Alu genomic architecture of SPAST predisposes to diverse CNV alleles with distinct transcriptional--and possibly phenotypic--consequences. Moreover, we provide further mechanistic insights into Alu-mediated copy-number change that are extendable to other loci.

Non-recurrent 17p11.2 deletions are generated by homologous and non-homologous mechanisms.

Several recurrent common chromosomal deletion and duplication breakpoints have been localized to large, highly homologous, low-copy repeats (LCRs). The mechanism responsible for these rearrangements, viz., non-allelic homologous recombination between LCR copies, has been well established. However, fewer studies have examined the mechanisms responsible for non-recurrent rearrangements with non-homologous breakpoint regions. Here, we have analyzed four uncommon deletions of 17p11.2, involving the Smith-Magenis syndrome region. Using somatic cell hybrid lines created from patient lymphoblasts, we have utilized a strategy based on the polymerase chain reaction to refine the deletion breakpoints and to obtain sequence data at the deletion junction. Our analyses have revealed that two of the four deletions are a product of Alu/Alu recombination, whereas the remaining two deletions result from a non-homologous end-joining mechanism. Of the breakpoints studied, three of eight are located in LCRs, and five of eight are within repetitive elements, including Alu and MER5B sequences. These findings suggest that higher-order genomic architecture, such as LCRs, and smaller repetitive sequences, such as Alu elements, can mediate chromosomal deletions via homologous and non-homologous mechanisms. These data further implicate homologous recombination as the predominant mechanism of deletion formation in this genomic interval.

Serial segmental duplications during primate evolution result in complex human genome architecture.

The human genome is particularly rich in low-copy repeats (LCRs) or segmental duplications (5%-10%), and this characteristic likely distinguishes us from lower mammals such as rodents. How and why the complex human genome architecture consisting of multiple LCRs has evolved remains an open question. Using molecular and computational analyses of human and primate genomic regions, we analyzed the structure and evolution of LCRs that resulted in complex architectural features of the human genome in proximal 17p. We found that multiple LCRs of different origins are situated adjacent to one another, whereas each LCR changed at different time points between >25 to 3-7 million years ago (Mya) during primate evolution. Evolutionary studies in primates suggested communication between the LCRs by gene conversion. The DNA transposable element MER1-Charlie3 and retroviral ERVL elements were identified at the breakpoint of the t(4;19) chromosome translocation in Gorilla gorilla, suggesting a potential role for transpositions in evolution of the primate genome. Thus, a series of consecutive segmental duplication events during primate evolution resulted in complex genome architecture in proximal 17p. Some of the more recent events led to the formation of novel genes that in human are expressed primarily in the brain. Our observations support the contention that serial segmental duplication events might have orchestrated primate evolution by the generation of novel fusion/fission genes as well as potentially by genomic inversions associated with decreased recombination rates facilitating gene divergence.

Mutations of RAI1, a PHD-containing protein, in nondeletion patients with Smith-Magenis syndrome.

Smith-Magenis syndrome (SMS) is a mental retardation/multiple congenital anomalies disorder associated with a heterozygous approximately 4-Mb deletion in 17p11.2. Patients with SMS show variability in clinical phenotype despite a common deletion found in >75-80% of patients. Recently, point mutations in the retinoic acid induced 1 (RAI1) gene, which lies within the SMS critical interval, were identified in three patients with many SMS features in whom no deletion was detected. It is not clear if the entire SMS phenotype can be accounted for by RAI1 haploinsufficiency, nor has the precise function of RAI1 been delineated. We report two novel RAI1 mutations, one frameshift and one nonsense allele, in nondeletion SMS patients. Comparisons of the clinical features in these two patients, three of the previously reported RAI1 point mutation cases, and the patients with a common deletion suggest that the majority of the clinical features in SMS result from RAI1 mutation, although phenotypic variability exists even among the individuals with RAI1 point mutations. Bioinformatics analyses of RAI1 and comparative genomics between human and mouse orthologues revealed a zinc finger-like plant homeo domain (PHD) at the carboxyl terminus that is conserved in the trithorax group of chromatin-based transcription regulators. These findings suggest RAI1 is involved in transcriptional control through a multi-protein complex whose function may be altered in individuals with SMS.

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.

Small marker chromosomes in two patients with segmental aneusomy for proximal 17p.

We report a nine-year-old girl (patient 1934) and a five-year-old boy (patient 2170) with small, de novo supernumerary marker chromosomes (SMCs) derived from proximal 17p. The clinical features of patient 1934 include developmental delay, triangular face, prominent forehead, low set ears, dental abnormalities, a high arched palate, long, flexible fingers, and joint laxity. Patient 2170 is affected with developmental delay, oral-motor dyspraxia/verbal apraxia, thick upper and lower lips, bilateral fifth finger clinodactyly, joint laxity and mild hypotonia. G-banded chromosome analysis of patient 1934 revealed mosaicism for a SMC in 72% of peripheral lymphocytes analyzed, whereas analysis of patient 2170 identified a smaller SMC present in 100% of cells analyzed. Fluorescence in situ hybridization (FISH) studies demonstrated that both of the SMCs derived from 17p10-p11.2. Using FISH and array-CGH analysis, the proximal breakpoints mapped within the centromere and the distal breakpoints were both located within the Smith-Magenis syndrome (SMS) common deletion region. We compare the clinical characteristics of our patients with those previously reported to have either SMC including 17p or duplications of proximal 17p in an effort to further delineate the phenotype of trisomy 17p10-p11.2 and to elucidate genotype-phenotype correlations.

Implications of human genome architecture for rearrangement-based disorders: the genomic basis of disease.

The term 'genomic disorder' refers to a disease that is caused by an alteration of the genome that results in complete loss, gain or disruption of the structural integrity of a dosage sensitive gene(s). In most of the common chromosome deletion/duplication syndromes, the rearranged genomic segments are flanked by large (usually >10 kb), highly homologous low copy repeat (LCR) structures that can act as recombination substrates. Recombination between non-allelic LCR copies, also known as non-allelic homologous recombination, can result in deletion or duplication of the intervening segment. Recent findings suggest that other chromosomal rearrangements, including reciprocal, Robertsonian and jumping translocations, inversions, isochromosomes and small marker chromosomes, may also involve susceptibility to rearrangement related to genome structure or architecture. In several cases, LCRs, AT-rich palindromes and pericentromeric repeats are located at such rearrangement breakpoints. Analysis of the products of recombination at the junctions of the rearrangements reveals both homologous recombination and non-homologous end joining as causative mechanisms. Thus, a more global concept of genomic disorders emerges in which susceptibility to rearrangements occurs due to underlying complex genomic architecture. Interestingly, this architecture plays a role not only in disease etiology, but also in primate genome evolution. In this review, we discuss recent advances regarding general mechanisms for the various rearrangements of our genome, and potential models for rearrangements with non-homologous breakpoint regions.

A girl with duplication 17p10-p12 associated with a dicentric chromosome.

We report a 7 1/2-year-old girl with an approximately 9.5 Mb duplication of proximal 17p. Her clinical features include moderately severe developmental delay, absence of speech, talipes, congenital dislocation of the hips, premature adrenarche, dysmorphic facial features, deep palmar creases, and signs and symptoms of peripheral neuropathy consistent with Charcot-Marie-Tooth disease type 1A (CMT1A). Chromosome analysis revealed a partially duplicated 17p with two centromeres on the derivative chromosome. Fluorescence in situ hybridization (FISH) analysis demonstrated the tandemly duplicated segment spans 17p10-p12, including the entire Smith-Magenis syndrome (SMS) critical region and a portion of the CMT1A critical region. One breakpoint mapped within the centromere and the second breakpoint mapped within the CMT1A critical region, distal to the PMP22 gene. Microsatellite polymorphism studies showed that the duplicated chromosome is of maternal origin. We compare the clinical features of our patient to those of individuals with partial trisomy of proximal 17p to further delineate the genotype-phenotype correlation associated with segmental duplication of this chromosomal region.

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.

Variability in clinical phenotype despite common chromosomal deletion in Smith-Magenis syndrome [del(17)(p11.2p11.2)].

PURPOSE: This report delineates the phenotypic features in a cohort of 58 individuals with Smith-Magenis syndrome (SMS) and compares features of patients with the common microdeletion to those of patients with variable sized deletions, and the three previously reported patients who harbor a mutation in RAI1 (retinoic acid induced 1). METHODS: From December 1990 thru September 1999, 58 persons with SMS were enrolled in a 5-day multidisciplinary clinical protocol at the General Clinical Research Center (GCRC), Texas Children's Hospital. Each patient had a cytogenetically evident deletion in 17p11.2. RESULTS: Of the 51 patients in whom the molecular extent of the chromosomal deletion could be delineated by pulsed-field gel electrophoresis (PFGE) and/or fluorescent in situ hybridization (FISH), 39 (approximately 76%) had the common SMS deletion. Smaller or larger deletions were seen in approximately 12% and approximately 10% of patients, respectively, and 1 patient had a complex chromosomal rearrangement including a deletion in 17p11.2. Parent of origin was determined by polymorphic marker analysis in a subset of patients: maternal approximately 43%, paternal approximately 57%. All patients had impaired cognitive and adaptive functioning and had at least one objective measure of sleep disturbance. Other common features (seen in >50% of patients) include short stature, ophthalmological, and otolaryngological anomalies, hearing impairment, abnormal EEG, and scoliosis. Cardiac and renal anomalies were seen in approximately 45% and approximately 19% of patients, respectively. There are no statistically significant differences in the incidence of these abnormalities in patients with the common deletion compared to those patients with smaller or larger sized deletions. CONCLUSIONS: Despite a common deletion size in 76% of patients with SMS, the only constant objectively defined features among these patients are sleep disturbances, low adaptive functioning, and mental retardation. There is no pathognomonic clinical feature, no characteristic cardiovascular defect, renal anomaly, otolaryngological or ophthalmic abnormality in SMS.

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.

Genetic proof of unequal meiotic crossovers in reciprocal deletion and duplication of 17p11.2.

A number of common contiguous gene syndromes have been shown to result from nonallelic homologous recombination (NAHR) within region-specific low-copy repeats (LCRs). The reciprocal duplications are predicted to occur at the same frequency; however, probably because of ascertainment bias and milder phenotypes, reciprocal events have been identified in only a few cases to date. We previously described seven patients with dup(17)(p11.2p11.2), the reciprocal of the Smith-Magenis syndrome (SMS) deletion, del(17)(p11.2p11.2). In >90% of patients with SMS, identical approximately 3.7-Mb deletions in 17p11.2 have been identified. These deletions are flanked by large (approximately 200 kb), highly homologous, directly oriented LCRs (i.e., proximal and distal SMS repeats [SMS-REPs]). The third (middle) SMS-REP is inverted with respect to them and maps inside the commonly deleted genomic region. To investigate the parental origin and to determine whether the common deletion and duplication arise by unequal crossovers mediated through NAHR between the proximal and distal SMS-REPs, we analyzed the haplotypes of 14 families with SMS and six families with dup(17)(p11.2p11.2), using microsatellite markers directly flanking the SMS common deletion breakpoints. Our data indicate that reciprocal deletion and duplication of 17p11.2 result from unequal meiotic crossovers. These rearrangements occur via both interchromosomal and intrachromosomal exchange events between the proximal and distal SMS-REPs, and there appears to be no parental-origin bias associated with common SMS deletions and the reciprocal duplications.