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.     

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.     

Inverted DNA repeats: a source of eukaryotic genomic instability.

While inverted DNA repeats are generally acknowledged to be an important source of genetic instability in prokaryotes, relatively little is known about their effects in eukaryotes. Using bacterial transposon Tn5 and its derivatives, we demonstrate that long inverted repeats also cause genetic instability leading to deletion in the yeast Saccharomyces cerevisiae. Furthermore, they induce homologous recombination. Replication plays a major role in the deletion formation. Deletions are stimulated by a mutation in the DNA polymerase delta gene (pol3). The majority of deletions result from imprecise excision between small (4- to 6-bp) repeats in a polar fashion, and they often generate quasipalindrome structures that subsequently may be highly unstable. Breakpoints are clustered near the ends of the long inverted repeats (< 150 bp). The repeats have both intra- and interchromosomal effects in that they also create hot spots for mitotic interchromosomal recombination. Intragenic recombination is 4 to 18 times more frequent for heteroalleles in which one of the two mutations is due to the insertion of a long inverted repeat, compared with other pairs of heteroalleles in which neither mutation has a long repeat. We propose that both deletion and recombination are the result of altered replication at the basal part of the stem formed by the inverted repeats.     

Alpha-galactosidase A gene rearrangements causing Fabry disease. Identification of short direct repeats at breakpoints in an Alu-rich gene

Fabry disease, an inborn error of glycosphingolipid catabolism, results from mutations in the X-linked gene encoding the lysosomal enzyme, alpha-galactosidase A (EC 3.2.1.22). Six alpha-galactosidase A gene rearrangements that cause Fabry disease were investigated to assess the role of Alu repetitive elements and short direct and/or inverted repeats in the generation of these germinal mutations. The breakpoints of five partial gene deletions and one partial gene duplication were determined by either cloning and sequencing the mutant gene from an affected hemizygote, or by polymerase chain reaction amplifying and sequencing the genomic region containing the novel junction. Although the alpha-galactosidase A gene contains 12 Alu repetitive elements (representing approximately 30% of the 12-kilobase (kb) gene or approximately 1 Alu/1.0 kb), only one deletion resulted from an Alu-Alu recombination. The remaining five rearrangements involved illegitimate recombinational events between short direct repeats of 2 to 6 base pairs (bp) at the deletion or duplication breakpoints. Of these rearrangements, one had a 3' short direct repeat within an Alu element, while another was unusual having two deletions of 1.7 kb and 14 bp separated by a 151-bp inverted sequence. These findings suggested that slipped mispairing or intrachromosomal exchanges involving short direct repeats were responsible for the generation of most of these gene rearrangements. There were no inverted repeat sequences or alternating purine-pyrimidine regions which may have predisposed the gene to these rearrangements. Intriguingly, the tetranucleotide CCAG and the trinucleotide CAG (or their respective complements, CTGG and CTG) occurred within or adjacent to the direct repeats at the 5' breakpoints in three and four of the five alpha-galactosidase A gene rearrangements, respectively, suggesting a possible functional role in these illegitimate recombinational events. These studies indicate that short direct repeats are important in the formation of gene rearrangements, even in human genes like alpha-galactosidase A that are rich in Alu repetitive elements.     

Characterization of the promoter controlling Mona/Gads expression in the megakaryocytic lineage

The deletion of a 260-kb segment containing all the coding DNA sequences (CDS) of chromosome 1 of Leishmania major Friedlin strain was performed through homologous recombination during a transfection experiment. This allowed the selection of a mutant clone containing a linear extra chromosome sizing 155 kb (XC155). The structure of XC155 was determined by restriction analysis and DNA cloning and sequencing of the gel-purified chromosome: it is made of a 'mirror' inverted duplication of the 'right' end of chromosome 1a (approximately 25 kb at each end), and in its central part of a complex tandem amplification of the linearized transfection vector containing the hygromycin resistance gene (over approximately 105 kb). No sequence of the coding region of chromosome 1 (including the 1.6-kb 'switch' region) was found. By contrast, XC155 contains two large (approximately 13 kb) clusters of tandemly repeated subtelomeric sequences (272-bp 'satellite' DNA) as well as telomeric hexamer repeats. This extra chromosome was found to be mitotically stable after >150 generations without selective pressure in vitro. Two sequence elements are considered which may have an effect on mitotic stability and participate to centromeric function in this extra chromosome: the amplification of the input vector and the 272-bp 'satellite' DNA bound by telomeric repeats.     

The NPHP1 gene deletion associated with juvenile nephronophthisis is present in a subset of individuals with Joubert syndrome

Joubert syndrome (JS) is an autosomal recessive multisystem disease characterized by cerebellar vermis hypoplasia with prominent superior cerebellar peduncles (the "molar tooth sign" [MTS] on axial magnetic resonance imaging), mental retardation, hypotonia, irregular breathing pattern, and eye-movement abnormalities. Some individuals with JS have retinal dystrophy and/or progressive renal failure characterized by nephronophthisis (NPHP). Thus far, no mutations in the known NPHP genes, particularly the homozygous deletion of NPHP1 at 2q13, have been identified in subjects with JS. A cohort of 25 subjects with JS and either renal and/or retinal complications and 2 subjects with only juvenile NPHP were screened for mutations in the NPHP1 gene by standard methods. Two siblings affected with a mild form of JS were found to have a homozygous deletion of the NPHP1 gene identical, by mapping, to that in subjects with NPHP alone. A control subject with NPHP and with a homozygous NPHP1 deletion was also identified, retrospectively, as having a mild MTS and borderline intelligence. The NPHP1 deletion represents the first molecular defect associated with JS in a subset of mildly affected subjects. Cerebellar malformations consistent with the MTS may be relatively common in patients with juvenile NPHP without classic symptoms of JS.     

Nonrandom Distribution of Chloroplast Recombination Events in Chlamydomonas Reinhardtii: Evidence for a Hotspot and an Adjacent Cold Region

Intermolecular recombination of Chlamydomonas chloroplast genes has been analyzed in sexual crosses and following biolistic transformation. The pattern and position of specific exchange events within 15 kb of the 22-kb inverted repeat have been mapped with respect to known restriction fragment length polymorphism markers that distinguish the chloroplast genomes of the interfertile species Chlamydomonas reinhardtii and Chlamydomonas smithii. Recombinant progeny were selected from two- and three-factor crosses involving point mutations conferring herbicide (dr) and antibiotic resistance (er and spr) in the psbA, 23S and 16S ribosomal RNA genes, respectively. Exchange events were not randomly distributed over the 15-kb region, but were found to occur preferentially in a 0.7-kb sequence spanning the 3' end of the psbA gene and were much less common in an adjacent region of ca. 2.0 kb. These findings are corroborated by data showing that the dr mutation is unlinked genetically (3% recombination/kb) to the er and spr rRNA mutations, which are themselves linked and show ca. 1% recombination/kb. This discrepancy is significant since the dr-er and er-spr intervals are about the same length (ca. 7 kb). During chloroplast transformation, the 0.7-kb recombination hotspot also functions as a preferential site for exchange events leading to the integration of donor psbA gene sequences. The 0.7-kb hotspot region contains four classes of 18-37-bp direct repeats also found in other intergenic regions, but no open reading frame. Using deletion constructs in a chloroplast transformation assay, the hotspot was localized to a 500-bp region that lacks most of these repeats, which suggests that the repeats themselves are not responsible for the increased recombination frequency. Within this region, a 400-bp sequence is highly conserved between the chloroplast genomes of C. reinhardtii and C. smithii and includes several structural motifs characteristic of recombination hotspots in other systems.     

Molecular characterization of CTNS deletions in nephropathic cystinosis: development of a PCR-based detection assay.

Nephropathic cystinosis is an autosomal recessive disorder that is characterized by accumulation of intralysosomal cystine and is caused by a defect in the transport of cystine across the lysosomal membrane. Using a positional cloning strategy, we recently cloned the causative gene, CTNS, and identified pathogenic mutations, including deletions, that span the cystinosis locus. Two types of deletions were detected-one of 9.5-16 kb, which was seen in a single family, and one of approximately 65 kb, which is the most frequent mutation found in the homozygous state in nearly one-third of cystinotic individuals. We present here characterization of the deletion breakpoints and demonstrate that, although both deletions occur in regions of repetitive sequences, they are the result of nonhomologous recombination. This type of mechanism suggests that the approximately 65-kb deletion is not a recurrent mutation, and our results confirm that it is identical in all patients. Haplotype analysis shows that this large deletion is due to a founder effect that occurred in a white individual and that probably arose in the middle of the first millenium. We also describe a rapid PCR-based assay that will accurately detect both homozygous and heterozygous deletions, and we use it to show that the approximately 65-kb deletion is present in either the homozygous or the heterozygous state in 76% of cystinotic patients of European origin.     

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.     

Structural plasmid instability in Bacillus subtilis: Effect of direct and inverted repeats

Summary Using precise excision as a model system, we have quantified the effect of direct repeats, inverted repeats and the size of the spacer between the repeats in the process of deletion formation in Bacillus subtilis. Both in the presence and absence of inverted repeats, the frequency of precise excision was strongly dependent on the direct repeat length. By increasing the direct repeat length from 9 bp to 18 and 27 bp, the precise excision frequency was raised by 3 and 4 orders of magnitude, respectively. In addition, irrespective of the direct repeat length, the presence of flanking inverted repeats enhanced the excision frequency by 3 orders of magnitude. Varying the inverted repeat length and the spacer size over a wide range did not significantly affect the excision frequencies. These results fit well into a model for deletion formation by slipped mispairing during replication of single-stranded plasmid DNA.     

A developmentally regulated deletion element with long terminal repeats has cis-acting sequences in the flanking DNA

Approximately 6000 specific DNA deletion events occur during development of the somatic macronucleus of the ciliate Tetrahymena. The eliminated Tlr1 element is 13 kb or more in length and has an 825 bp inverted repeat near the rearrangement junctions. A functional analysis of the cis-acting sequences required for Tlr1 rearrangement was performed. A construct consisting of the entire inverted repeat and several hundred base pairs of flanking DNA on each side was rearranged accurately in vivo and displayed junctional variability similar to the chromosomal Tlr1 rearrangement. Thus, 11 kb or more of internal element DNA is not required in cis for DNA rearrangement. A second construct with only 51 bp of Tetrahymena DNA flanking the right junction underwent aberrant rearrangement. Thus, a signal for determination of the Tlr1 junction is located in the flanking DNA, 51 bp or more from the right junction. Within the Tlr1 inverted repeat are 19 bp tandem repeats. A construct with the 19mer repeat region deleted from the right half of the inverted repeat utilized normal rearrangement junctions. Thus, despite its transposon-like structure, Tlr1 is similar to other DNA rearrangements in Tetrahymena in possessing cis-acting sequences outside the deleted DNA.     

Mucopolysaccharidosis type IVA: common double deletion in the N-acetylgalactosamine-6-sulfatase gene (GALNS)

Mucopolysaccharidosis IVA (MPS IVA) is an autosomal recessive disorder caused by a deficiency in N-acetylgalactosamine-6-sulfatase (GALNS). We found two separate deletions of nearly 8.0 and 6.0 kb in the GALNS gene, including some exons. There are Alu repetitive elements near the breakpoints of the 8.0-kb deletion, and this deletion resulted from an Alu—Alu recombination. The other 6.0-kb deletion involved illegitimate recombinational events between incomplete short direct repeats of 8 bp at deletion breakpoints. The same rearrangement has been observed in a heteroallelic state in four unrelated patients. This is the first documentation of a common double deletion a gene that is not a member of a gene cluster.     

Low-copy repeats mediate the common 3-Mb deletion in patients with velo-cardio-facial syndrome

Velo-cardio-facial syndrome (VCFS) is the most common microdeletion syndrome in humans. It occurs with an estimated frequency of 1 in 4, 000 live births. Most cases occur sporadically, indicating that the deletion is recurrent in the population. More than 90% of patients with VCFS and a 22q11 deletion have a similar 3-Mb hemizygous deletion, suggesting that sequences at the breakpoints confer susceptibility to rearrangements. To define the region containing the chromosome breakpoints, we constructed an 8-kb-resolution physical map. We identified a low-copy repeat in the vicinity of both breakpoints. A set of genetic markers were integrated into the physical map to determine whether the deletions occur within the repeat. Haplotype analysis with genetic markers that flank the repeats showed that most patients with VCFS had deletion breakpoints in the repeat. Within the repeat is a 200-kb duplication of sequences, including a tandem repeat of genes/pseudogenes, surrounding the breakpoints. The genes in the repeat are GGT, BCRL, V7-rel, POM121-like, and GGT-rel. Physical mapping and genomic fingerprint analysis showed that the repeats are virtually identical in the 200-kb region, suggesting that the deletion is mediated by homologous recombination. Examination of two three-generation families showed that meiotic intrachromosomal recombination mediated the deletion.     

Identification of a 3.0-kb major recombination hotspot in patients with Sotos syndrome who carry a common 1.9-Mb microdeletion

Sotos syndrome (SoS) is a congenital dysmorphic disorder characterized by overgrowth in childhood, distinctive craniofacial features, and mental retardation. Haploinsufficiency of the NSD1 gene owing to either intragenic mutations or microdeletions is known to be the major cause of SoS. The common approximately 2.2-Mb microdeletion encompasses the whole NSD1 gene and neighboring genes and is flanked by low-copy repeats (LCRs). Here, we report the identification of a 3.0-kb major recombination hotspot within these LCRs, in which we mapped deletion breakpoints in 78.7% (37/47) of patients with SoS who carry the common microdeletion. The deletion size was subsequently refined to 1.9 Mb. Sequencing of breakpoint fragments from all 37 patients revealed junctions between a segment of the proximal LCR (PLCR-B) and the corresponding region of the distal LCR (DLCR-2B). PLCR-B and DLCR-2B are the only directly oriented regions, whereas the remaining regions of the PLCR and DLCR are in inverted orientation. The PLCR, with a size of 394.0 kb, and the DLCR, with a size of of 429.8 kb, showed high overall homology (approximately 98.5%), with an increased sequence similarity (approximately 99.4%) within the 3.0-kb breakpoint cluster. Several recombination-associated motifs were identified in the hotspot and/or its vicinity. Interestingly, a 10-fold average increase of a translin motif, as compared with the normal distribution within the LCRs, was recognized. Furthermore, a heterozygous inversion of the interval between the LCRs was detected in all fathers of the children carrying a deletion in the paternally derived chromosome. The functional significance of these findings remains to be elucidated. Segmental duplications of the primate genome play a major role in chromosomal evolution. Evolutionary study showed that the duplication of the SoS LCRs occurred 23.3-47.6 million years ago, before the divergence of Old World monkeys.     

Ac Induces Homologous Recombination at the Maize P Locus

The maize P gene conditions red phlobaphene pigmentation to the pericarp and cob. Starting from two unstable P alleles which carry insertions of the transposable element Ac, we have derived 51 P null alleles; 47 of the 51 null alleles have a 17-kb deletion which removes the 4.5-kb Ac element and 12.5 kb of P sequences flanking both sides of Ac. The deletion endpoints lie within two 5.2-kb homologous direct repeats which flank the P gene. A P allele which contains the direct repeats, but does not have an Ac insertion between the direct repeats, shows very little sporophytic or gametophytic instability. The apparent frequency of sporophytic mutations was not increased when Ac was introduced in trans. Southern analysis of DNA prepared from the pericarp tissue demonstrates that the deletions can occur premeiotically, in the somatic cells during development of the pericarp. Evidence is presented that the deletions occurred by homologous recombination between the two direct repeats, and that the presence of an Ac element at the P locus is associated with the recombination/deletion. These results add another aspect to the spectrum of activities of Ac: the destabilization of flanking direct repeat sequences.     

Chromosomal arm replacement in Streptomyces griseus

UV irradiation of Streptomyces griseus 2247 yielded a new chromosomal deletion mutant, MM9. Restriction and sequencing analysis revealed that homologous recombination between two similar lipoprotein-like open reading frames, which are located 450 and 250 kb from the left and right ends, respectively, caused chromosomal arm replacement. As a result, new 450-kb terminal inverted repeats (TIRs) were formed in place of the original 24-kb TIRs. Frequent homologous recombinations in Streptomyces strains suggest that telomere deletions can usually be repaired by recombinational DNA repair functioning between the intact and deleted TIR sequences on the same chromosome.     

Human meiotic recombination products revealed by sequencing a hotspot for homologous strand exchange in multiple HNPP deletion patients.

The HNPP (hereditary neuropathy with liability to pressure palsies) deletion and CMT1A (Charcot-Marie-Tooth disease type 1A) duplication are the reciprocal products of homologous recombination events between misaligned flanking CMT1A-REP repeats on chromosome 17p11. 2-p12. A 1.7-kb hotspot for homologous recombination was previously identified wherein the relative risk of an exchange event is 50 times higher than in the surrounding 98.7% identical sequence shared by the CMT1A-REPs. To refine the region of exchange further, we designed a PCR strategy to amplify the recombinant CMT1A-REP from HNPP patients as well as the proximal and distal CMT1A-REPs from control individuals. By comparing the sequences across recombinant CMT1A-REPs to that of the proximal and distal CMT1A-REPs, the exchange was mapped to a 557-bp region within the previously identified 1.7-kb hotspot in 21 of 23 unrelated HNPP deletion patients. Two patients had recombined sequences suggesting an exchange event closer to the mariner-like element previously identified near the hotspot. Five individuals also had interspersed patches of proximal or distal repeat specific DNA sequence indicating potential gene conversion during the exchange of genetic material. Our studies provide a direct observation of human meiotic recombination products. These results are consistent with the hypothesis that minimum efficient processing segments, which have been characterized in Escherichia coli, yeast, and cultured mammalian cells, may be required for efficient homologous meiotic recombination in humans.     

Different mechanisms inferred from sequences of human mitochondrial DNA deletions in ocular myopathies.

We have sequenced the deletion borders of the muscle mitochondrial DNA from 24 patients with heteroplasmic deletions. The length of these deletions varies from 2.310 bp to 8.476 bp and spans from position 5.786 to 15.925 of the human mitochondrial genome preserving the heavy chain and light chain origins of replication. 12 cases are common deletions identical to the mutation already described by other workers and characterized by 13 bp repeats at the deletion boundaries, one of these repeats being retained during the deletion process. The other cases (10 out of 12) have shown deletions which have not been previously described. All these deletions are located in the H strand DNA region which is potentially single stranded during mitochondrial DNA replication. In two cases, the retained Adenosine from repeat closed to the heavy strand origin of replication would indicate slippage mispairing. Furthermore in one patient two mt DNA molecules have been cloned and their sequences showed the difference of four nucleotides in the breakpoint of the deletion, possibly dued to slippage mispairing. Taken together our results suggest that deletions occur either by slippage mispairing or by internal recombination at the direct repeat level. They also suggest that different mechanisms account for the deletions since similarly located deletions may display different motives at the boundaries including the absence of any direct repeat.     

Short direct repeats at the breakpoints of a novel large deletion in the CFTR gene suggest a likely slipped mispairing mechanism

In the cystic fibrosis conductance transmembrane regulator (CFTR) gene a few small deletions and only a large, complex, 50-kb deletion have been described so far. We report a second large deletion, which had been hypothesized in a patient affected by cystic fibrosis on the basis of an abnormal pattern of inheritance of the intragenic microsatellites IVS17b/TA and IVS17b/CA. Southern blot analysis revealed the presence of an anomalous band in the patient and her father, in the region encompassing exons 13 – 19, approximately 0.6 kb shorten than the one present in normal controls, in addition to the band of the correct size. Cloning and sequencing the DNA fragments spanning the region of interest demonstrated the presence of a 703-bp deletion causing complete removal of exon 17b in the paternal cystic fibrosis chromosome. This analysis revealed the presence of two short direct repeats flanking the breakpoints. The 3′ repeat partially overlapped the IVS17b/CA microsatellite and the number of CA repeated units present in the paternal cystic fibrosis allele was the shortest ever found among chromosomes so far analyzed. These data may suggest that the mechanism for the generation of the deletion may have involved a slipped mispairing during DNA replication, which has not previously been described in the CFTR gene. Received: 27 December 1995 / Accepted: 30 January 1996     

De novo rearrangements found in 2% of index patients with spinal muscular atrophy: mutational mechanisms, parental origin, mutation rate, and implications for genetic counseling.

Spinal muscular atrophy (SMA) is a relatively common autosomal recessive neuromuscular disorder. We have identified de novo rearrangements in 7 (approximately 2%) index patients from 340 informative SMA families. In each, the rearrangements resulted in the absence of the telomeric copy of the survival motor neuron (SMN) gene (telSMN), in two cases accompanied by the loss of the neuronal apoptosis-inhibitory protein gene . Haplotype analysis revealed unequal recombination in four cases, with loss of markers Ag1-CA and C212, which are near the 5' ends of the SMN genes. In one case, an interchromosomal rearrangement involving both the SMN genes and a regrouping of Ag1-CA and C212 alleles must have occurred, suggesting either interchromosomal gene conversion or double recombination. In two cases, no such rearrangement was observed, but loss of telSMN plus Ag1-CA and C212 alleles in one case suggested intrachromosomal deletion or gene conversion. In six of the seven cases, the de novo rearrangement had occurred during paternal meiosis. Direct detection of de novo SMA mutations by molecular genetic means has allowed us to estimate for the first time the mutation rate for a recessive disorder in humans. The sex-averaged rate of 1.1 x 10(-4), arrived at in a proband-based approach, compares well with the rate of 0.9 x 10(-4) expected under a mutation-selection equilibrium for SMA. These findings have important implications for genetic counseling and prenatal diagnosis in that they emphasize the relevance of indirect genotype analysis in combination with direct SMN-gene deletion testing in SMA families.