Alu-mediated Homologous Recombination Research Articles

Genetic mutation spectrum of pantothenate kinase-associated neurodegeneration expanded by breakpoint sequencing in pantothenate kinase 2 gene

BackgroundNeurodegeneration with brain iron accumulation describes a group of rare heterogeneous progressive neurodegenerative disorders characterized by excessive iron accumulation in the basal ganglia region. Pantothenate kinase-associated neurodegeneration (PKAN) is a major form of this disease.ResultsA total of 7 unrelated patients were diagnosed with PKAN in a single tertiary center from August 2009 to February 2018. Ten variants in PANK2 including three novel sequence variants and one large exonic deletion were detected. Sequencing of the breakpoint was performed to predict the mechanism of large deletion and AluSx3 and AluSz6 were found with approximately 97.3% sequence homology.ConclusionThe findings support the disease-causing role of PANK2 and indicate the possibility that exonic deletion of PANK2 found in PKAN is mediated through Alu-mediated homologous recombination.

Real-time PCR-based detection of the Alu-mediated deletion of FUT2 (sedel2).

Secretor status of the ABH(O) histoblood group antigens is regulated by secretor type α(1,2)fucosyltransferase encoded by FUT2. The sedel2 allele is a complete deletion of the FUT2 coding region generated by Alu-mediated homologous recombination. This deletion seems to be exclusively encountered in certain Oceanian populations. From the perspective of forensic science, sedel2 is considered to be one of ancestry informative markers for these populations. Real-time PCR followed by melting curve analysis was employed to find primer set to specifically amplify sedel2. We designed primers which produced a 231-bp amplicon specific to sedel2. The specificity of these primers was also confirmed by gel electrophoresis and sequencing of the PCR product. Then, two real-time PCR methods based on melting curve analysis and a hydrolysis probe were designed to determine sedel2 zygosity by adding FUT2-specific primers. These two methods were validated by analyzing 24 Samoan subjects. The results obtained from 24 Samoan subjects by the two methods were fully in accordance with those obtained by a previous conventional PCR method that amplified a 2.7-kb fragment of sedel2. Therefore, these two methods seemed to accurately determine the zygosity of sedel2 and were useful for investigation of the distribution and origin of this deletion.

Pathogenicity reclassification of two BRCA1/BRCA2 exonic duplications after identification of genomic breakpoints and tandem orientation

The genomic consequence and clinical interpretation of large duplications are difficult to infer without determining the location and orientation of the duplicated sequence. We aimed to characterize two intragenic duplications detected in two hereditary breast and ovarian cancer syndrome (HBOC) families, namely BRCA1 exon 4 to 6 and BRCA2 exon 17 to 18, previously detected by multiplex ligation probe amplification and initially classified as variants of unknown significance. Using long range PCR, with duplication-specific primers, we were able to ascertain the genomic breakpoints and observed that the two rearrangements occurred in tandem and in direct orientation. The BRCA1 c.134+440_441+870dup and BRCA2 c.7806-2083_8332-1512dup duplications here identified are predicted to cause frameshifts that create a premature stop codon and were reclassified as pathogenic. Furthermore, both families present phenotypic traits typical of HBOC syndrome. We also observed that the genomic breakpoints of these two duplications occurred within highly homologous Alu elements. Concluding, we characterized two in tandem BRCA1 and BRCA2 duplications that likely occurred by Alu-mediated homologous recombination, allowing identification of the underlying cause of the HBOC syndrome in these families.

Nebulin: A Study of Protein Repeat Evolution

Protein domain repeats are common in proteins that are central to the organization of a cell, in particular in eukaryotes. They are known to evolve through internal tandem duplications. However, the understanding of the underlying mechanisms is incomplete. To shed light on repeat expansion mechanisms, we have studied the evolution of the muscle protein Nebulin, a protein that contains a large number of actin-binding nebulin domains. Nebulin proteins have evolved from an invertebrate precursor containing two nebulin domains. Repeat regions have expanded through duplications of single domains, as well as duplications of a super repeat (SR) consisting of seven nebulins. We show that the SR has evolved independently into large regions in at least three instances: twice in the invertebrate Branchiostoma floridae and once in vertebrates. In-depth analysis reveals several recent tandem duplications in the Nebulin gene. The events involve both single-domain and multidomain SR units or several SR units. There are single events, but frequently the same unit is duplicated multiple times. For instance, an ancestor of human and chimpanzee underwent two tandem duplications. The duplication junction coincides with an Alu transposon, thus suggesting duplication through Alu-mediated homologous recombination. Duplications in the SR region consistently involve multiples of seven domains. However, the exact unit that is duplicated varies both between species and within species. Thus, multiple tandem duplications of the same motif did not create the large Nebulin protein. Finally, analysis of segmental duplications in the human genome reveals that duplications are more common in genes containing domain repeats than in those coding for nonrepeated proteins. In fact, segmental duplications are found three to six times more often in long repeated genes than expected by chance.

Characterization of a Novel Alu-Alu Recombination-Mediated Genomic Deletion in the TCIRG1 Gene in Five Osteopetrotic Patients

Human malignant autosomal recessive osteopetrosis (ARO) is a genetically heterogeneous disorder caused by reduced bone resorption by osteoclasts. Biallelic mutations in the TCIRG1 gene, encoding the a3 subunit of the vacuolar proton pump, are responsible for more than one half of ARO patients. However, a few patients with monoallelic mutations have been described, raising the possibility of a dominant-like TCIRG1-dependent osteopetrosis, of a digenic disease, or of peculiar mutations difficult to detect with standard methods. We describe here a novel genomic deletion in the TCIRG1 gene explaining why, in some patients, mutations in only one allele have previously been found. The analysis of a proband from a consanguineous Turkish family allowed us to define the deletion boundaries encompassing introns 10 and 13 and occurring within AluSx repeat sequences, suggesting Alu-mediated homologous recombination as a mechanism. An identical genomic deletion at the heterozygous level was found in four unrelated Italian families in whom only a single mutated allele has previously been found. TCIRG1 haplotype analysis in these five families suggests a possible common ancestral origin for this large deletion. In summary, we describe the identification of a novel genomic deletion in the TCIRG1 gene that is of clinical relevance, especially in prenatal diagnosis.

Molecular Basis of Kindler Syndrome in Italy: Novel and Recurrent Alu/Alu Recombination, Splice Site, Nonsense, and Frameshift Mutations in the KIND1 Gene

Kindler syndrome (KS) is a rare autosomal recessive disorder characterized by skin blistering in childhood followed by photosensitivity and progressive poikiloderma. Most cases of KS result from mutations in the KIND1 gene encoding kindlin-1, a component of focal adhesions in keratinocytes. Here, we report novel and recurrent KIND1 gene mutations in nine unrelated Italian KS individuals. A novel genomic deletion of approximately 3.9 kb was identified in four patients originating from the same Italian region. This mutation deletes exons 10 and 11 from the KIND1 mRNA leading to a truncated kindlin-1. The deletion breakpoint was embedded in AluSx repeats, specifically in identical 30-bp sequences, suggesting Alu-mediated homologous recombination as the pathogenic mechanism. KIND1 haplotype analysis demonstrated that patients with this large deletion were ancestrally related. Five additional mutations were disclosed, two of which were novel. To date, four recurrent mutations have been identified in Italian patients accounting for approximately approximately 75% of KS alleles in this population. The abundance of repetitive elements in intronic regions of KIND1, together with the identification of a large deletion, suggests that genomic rearrangements could be responsible for a significant proportion of KS cases. This finding has implications for optimal KIND1 mutational screening in KS individuals.

Screening for germline rearrangements in BRCA1 and BRCA2in Norwegian families with breast or breast/ovarian cancer

Standard PCR-based mutation detection strategies performed on the BRCA1 and BRCA2 genes of breast and ovarian cancer families are mostly aimed at identifying changes in the coding sequences and in the donor-acceptor splice sites. Hence, mutations in the promoter and the untranslated regions, and large rearrangements, are not detected by these methods. To assess the importance of BRCA1 and BRCA2 alterations that are neglected by standard screening methods, we monitored germline rearrangements in these genes using 'multiplex ligation-dependent probe amplification' technology [1]. One hundred and seventy-nine Norwegian breast and ovarian cancer families were screened for rearrangements in BRCA1 while 97 families were tested for aberrations in BRCA2. Whereas no rearrangements were detected in BRCA2, four distinct deletions were found in BRCA1. Those deletions originating by Alu-mediated homologous recombination include: exons 1–13, exons 3–16, exons 8–13 and exon 23, respectively. The large 23.8 kb deletion excluding exons 8–13 in BRCA1 has been found both in the French and British breast cancer population [2-4]. The deletions of exons 1–13, exons 3–16 and exon 23 have not been previously reported.

Identification and characterization of novel mutations of the major Fanconi anemia gene FANCA in the Japanese population

Fanconi anemia (FA) is a rare autosomal recessive disorder of hematopoiesis, with at least 11 complementation groups. FANCA, a gene for group A, accounts for the majority of FA patients. Previous studies of FANCA mutations revealed high allelic heterogeneity, frequent occurrence of large deletions, and interpopulation differences. However, systematic mutational analysis, including gene dosage assay to detect large deletions, has not been documented for Asian populations. A newly developed TaqMan quantitative PCR-based gene dosage assay, combined with sequencing of exons and cDNA fragments, allowed for detection of 48 mutant alleles of FANCA in 27 (77%) of 35 unrelated Japanese FA families with no detectable mutations in FANCC or FANCG. We identified 29 different mutations (21 nucleotide substitutions or small deletions/insertions and eight large deletions), at least 20 of which were novel. The FANCA mutational spectrum of the Japanese was different from that of other ethnic groups so far studied. This is the largest scale of mutation analysis of FANCA in the Japanese population. Characterization of these mutations provided new information regarding the mutagenesis mechanisms and structure-function relationship of FANCA. Specifically, our data suggest that diverse mechanisms including nonhomologous recombination as well as Alu-mediated homologous recombination are involved in the generation of large deletions in FANCA.

Homeologous recombination between AluSx-sequences as a cause of hemophilia.

Although large deletions from the coagulation factor VIII gene, F8, are responsible for 5% of severe hemophilia A (seHA), few of them have been fully characterised. A detailed description of a large partial deletion of the F8 caused by unequal recombination between homeologous AluSx-derived sequences is presented. The proband, a case of isolated hemophilia A with a high inhibitor titre (5700 BU), showed a consistent absence of PCR-amplification of exons 4 to 10, EX4_EX10del. Two approaches were used to narrow down the deletion breakpoints: a direct physical analysis based on PCR (that additionally permits carrier detection in the family); and, under the hypothesis that the mutation resulted from homologous recombination, sequence alignments of F8 intron 3 and 10. Both approaches indicate an unequal crossing over (CO) between two Alu-related sequences. Both elements involved were derived from the AluSx-subfamily consensus and demonstrate 86% sequence identity (with only single-base mismatches), with three gaps (of 2, 3 and 14-bases) and two main tracts of perfectly homologous sequence (28 and 24-bp). The short stretch of intron 10 embedded into intron 3 sequence, linked to the CO, represents a typical hallmark of homologous recombination (double-strand break repair model). A detailed description of EX4_EX10del mutation is c.[338+3485delins1687+2223_1687+2225; 338+3551_1687+2291 del]. The common involvement of unequal homologous recombination mediated by repetitive elements allowed us to suggest that our experimental design (based on intron sequence alignments) may be successfully applied to rearrangements involved in other X-linked inherited diseases. Like other Alu-rich genes throughout the human genome, Alu-mediated homologous recombination in F8 may be an important cause of hemophilia by promoting large DNA deletions.

Different molecular mechanisms underlie genomic deletions in the MLH1 Gene.

In this study we examined a series of 52 patients belonging to hereditary nonpolyposis colorectal cancer (HNPCC) or HNPCC-related families, all who had previously tested negative for mismatch repair (MMR) gene point mutations. Southern blot mutational screening of MLH1 and MSH2 genes was carried out with the aim of detecting large genomic rearrangements and of identifying the molecular mechanisms underlying the inactivation of the MMR genes. Three patients had abnormal restriction patterns and were found to carry distinct MLH1 internal deletions. Long-range PCRs identified the loss of DNA tracts spanning exon 6 (about 2.4 kb in proband A-AV20 and 0.8 kb in proband A-PD5) and exon 3 (about 2.5 kb in proband R-RM2). In A-AV20 the breakpoints occurred into identical 33-bp regions in introns 5 and 6 and a mechanism of classical Alu-mediated homologous recombination was evident. Also, in patient A-PD5 the breakpoints were located in these introns, but without direct involvement of repetitive sequences. In patient R-RM2 the breakpoints were located within repetitive L1 elements with poor homology in intron 2 and 3 and the rearranged allele was characterized by a complex insertion deletion (delCCinsACATAGTA), giving rise to a palindromic CTTAACATAGTATGTTAAG sequence in proximity of the fusion site. This study confirms that genomic rearrangements are an important component of the spectrum of MMR mutations. Although Alu repeats are likely to be implicated in the majority of cases, different molecular mechanisms may also be responsible for the observed MLH1 intragenic deletions. In particular, HNPCC resulting from L1-mediated recombination has been identified as a novel mechanism for MMR inactivating mutation.

Molecular description of three macro-deletions and an Alu–Alu recombination-mediated duplication in the HPRT gene in four patients with Lesch-Nyhan disease

Mutations in the HPRT gene cause a spectrum of diseases that ranges from hyperuricemia alone to hyperuricemia with profound neurological and behavioral dysfunction. The extreme phenotype is termed Lesch-Nyhan syndrome. In 271 cases in which the germinal HPRT mutation has been characterized, 218 different mutations have been found. Of these, 34 (13%) are large- (macro-) deletions of one exon or greater and four (2%) are partial gene duplications. The deletion breakpoint junctions have been defined for only three of the 34 macro-deletions. The molecular basis of two of the four duplications has been defined. We report here the breakpoint junctions for three new deletion mutations, encompassing exons 4–8 (20 033 bp), exons 4 and 5 (13 307 bp) and exons 5 and 6 (9454 bp), respectively. The deletion breakpoints were defined by a combination of long polymerase chain reaction (PCR) amplifications, and conventional PCR and DNA sequencing. All three deletions are the result of non-homologous recombinations. A fourth mutation, a duplication of exons 2 and 3, is the result of an Alu-mediated homologous recombination between identical 19 bp sequences in introns 3 and 1. In toto, two of three germinal HPRT duplication mutations appear to have been caused by Alu-mediated homologous recombination, while only one of six deletion mutations appears to have resulted from this type of recombination mechanism. The other five deletion mutations resulted from non-homologous recombination. With this admittedly limited number of characterized macro-mutations, Alu-mediated unequal homologous recombinations account for at least 8% (3 of 38) of the macro-alterations and 1% (3 of 271) of the total HPRT germinal mutations.

Compound Heterozygosity for a Recurrent 16.5-kb Alu-Mediated Deletion Mutation and Single-Base-Pair Substitutions in the ABCC6 Gene Results in Pseudoxanthoma Elasticum

Pseudoxanthoma elasticum (PXE) is a systemic heritable disorder affecting the elastic structures in the skin, eyes, and cardiovascular system, with considerable morbidity and mortality. Recently, mutations in the ABCC6 gene (also referred to as "MRP6" or "eMOAT") encoding multidrug-resistance protein 6 (MRP6), a putative transmembrane ABC transporter protein of unknown function, have been disclosed. Most of the genetic lesions delineated thus far consist of single-base-pair substitutions resulting in nonsense, missense, or splice-site mutations. In this study, we examined four multiplex families with PXE inherited in an autosomal recessive pattern. In each family, the proband was a compound heterozygote for a single-base-pair-substitution mutation and a novel, approximately 16.5-kb deletion mutation spanning the site of the single-base-pair substitution in trans. The deletion mutation was shown to extend from intron 22 to intron 29, resulting in out-of-frame deletion of 1,213 nucleotides from the corresponding mRNA and causing elimination of 505 amino acids from the MRP6 polypeptide. The deletion breakpoints were precisely the same in all four families, which were of different ethnic backgrounds, and haplotype analysis by 13 microsatellite markers suggested that the deletion had occurred independently. Deletion breakpoints within introns 22 and 29 were embedded within AluSx repeat sequences, specifically in a 16-bp segment of DNA, suggesting Alu-mediated homologous recombination as a mechanism.

In-frame deletions of BRCA1 may define critical functional domains

The identification of genomic rearrangements involving more than 0.5 kb of the BRCA1 gene has confirmed a more complex mutation spectrum than was initially appreciated. Genomic rearrangements in BRCA1 represent 15% of all mutations in a group of French and American breast and ovarian cancer families and 36% of all mutations in a group of Dutch families. The rearrangements described to date range in size from 510 bp to 23.8 kb, are found throughout the gene, and are most frequently attributable to homologous recombination. We describe the identification of rearrangements in two breast and ovarian cancer families that involve 3.4 and 11.5 kb of the BRCA1 gene and span multiple exons but maintain the reading frame. Both gene rearrangements appear to result from Alu-mediated homologous recombination and have been detected by using a combination of protein truncation analysis and Southern blot analysis. These rearrangements result in the loss of amino acids that lie at the carboxy-terminus of the protein and that have previously been shown to have functional significance. Because these rearrangements result in the deletion of exons but maintain the reading frame, they may provide insights into specific regions and amino acids that have functional significance for the BRCA1 protein.

Structure of theMLT gene and molecular characterization of the genomic breakpoint junctions in the t(11;18)(q21;q21) of marginal zone B-cell lymphomas of MALT type

The t(11;18)(q21;q21) between the inhibitor of apoptosis API2 and the MLT gene is a distinct feature of marginal zone B-cell lymphomas of MALT-type. Hitherto the chimeric API2-MLT transcripts are all "in-frame" and predominantly fuse exon 7 of API2 to different MLT exons. Recurrent chromosomal translocations are common in lymphoid neoplasms and might represent by-products of the rearrangement processes generating antigen receptor diversity. The genomic structure of the MLT gene was determined to facilitate amplification of the genomic breakpoint junctions from 5 MALT-type lymphomas with t(11;18). Their sequence analysis showed scattering of the chromosome 11 breakpoints in intron 7 of API2 whereas rearrangements in MLT occurred in intron 2, 4, 7, or 8, respectively. Sequences around the junctions did not display recognition signal sequences mediating lymphocytic V(D)J recombination or other sequence motifs associated with recombination. The breakpoints occurred in a copy of an AluSx repeat in three cases, but interchromosomal Alu-mediated homologous recombination could be ruled out as the repeat resided only on one of the participating chromosomes. The t(11;18) was associated with a deletion in 4 out of 5 cases, ranging in size from 53 bp up to more than 200 kb. These deletions were observed on one or sometimes both derivative chromosomes that might indicate the susceptibility of these regions for breakage. Our data suggest that the API2-MLT fusion might result from a non-homologous end joining event after multiple double-strand breaks. The clustering of breaks in intron 7 of API2 and the consistent "in frame" API2-MLT fusions could therefore reflect certain functional constraints crucial for clonal outgrowth.

Analysis of the region of the 5′ end of the MLL gene involved in genomic duplication events

Rearrangements of the MLL gene are associated with both myeloid and lymphoid acute leukaemia. The gene is commonly involved in reciprocal translocations leading to the creation of chimaeric genes encoding novel protein products. An alternative mechanism of MLL gene rearrangement is due to intragenic duplication, leading to partial duplication of the amino-terminal portion of the protein. This occurs in leukaemia, but it has recently been shown that partial duplications of the MLL gene are detectable in peripheral blood and bone marrow of healthy donors and in normal non-haemopoietic tissues. Sequence analysis of the 45 kb of the 5' end of the MLL locus encompassing the breakpoints of these genomic duplications has failed to show a definitive reason as to why this region is such a frequent target of rearrangement. Indeed, although the majority of the breakpoint joins are the result of apparent Alu-mediated homologous recombination, several joins do not involve Alu elements in the region, despite a high density of these repetitive elements in the sequence.

Analysis of the region of the 5' end of the MLL gene involved in genomic duplication events

Rearrangements of the MLL gene are associated with both myeloid and lymphoid acute leukaemia. The gene is commonly involved in reciprocal translocations leading to the creation of chimaeric genes encoding novel protein products. An alternative mechanism of MLL gene rearrangement is due to intragenic duplication, leading to partial duplication of the amino-terminal portion of the protein. This occurs in leukaemia, but it has recently been shown that partial duplications of the MLL gene are detectable in peripheral blood and bone marrow of healthy donors and in normal non-haemopoietic tissues. Sequence analysis of the 45 kb of the 5′ end of the MLL locus encompassing the breakpoints of these genomic duplications has failed to show a definitive reason as to why this region is such a frequent target of rearrangement. Indeed, although the majority of the breakpoint joins are the result of apparent Alu-mediated homologous recombination, several joins do not involve Alu elements in the region, despite a high density of these repetitive elements in the sequence.