Exact breakpoints of In(1)wm4 rearrangement

In(1)wm4 was known for decades as a classic example of position effect variegation-causing rearrangement and was mentioned in hundreds of publications. Nevertheless, the euchromatin breakpoint position of the rearrangement was not precisely localized. We performed nanopore sequencing of DNA from In(1)wm4 homozygous flies and determined the exact position of euchromatic (chrX:2767875) and heterochromatic breakpoints of the rearrangement.

Fine Breakpoint Mapping by Genome Sequencing Reveals the First Large X Inversion Disrupting the NHS Gene in a Patient with Syndromic Cataracts

Inversions are structural variants that are generally balanced. However, they could lead to gene disruptions or have positional effects leading to diseases. Mutations in the NHS gene cause Nance-Horan syndrome, an X-linked disorder characterised by congenital cataracts and dental anomalies. Here, we aimed to characterise a balanced pericentric inversion X(p22q27), maternally inherited, in a child with syndromic bilateral cataracts by breakpoint mapping using whole-genome sequencing (WGS). 30× Illumina paired-end WGS was performed in the proband, and breakpoints were confirmed by Sanger sequencing. EdU assays and FISH analysis were used to assess skewed X-inactivation patterns. RNA expression of involved genes in the breakpoint boundaries was evaluated by droplet-digital PCR. We defined the breakpoint position of the inversion at Xp22.13, with a 15 bp deletion, disrupting the unusually large intron 1 of the canonical NHS isoform, and also perturbing topologically-associated domains (TADs). Moreover, a microhomology region of 5 bp was found on both sides. RNA analysis confirmed null and reduced NHS expression in the proband and his unaffected mother, respectively. In conclusion, we report the first chromosomal inversion disrupting NHS, fine-mapped by WGS. Our data expand the clinical spectrum and the pathogenic mechanisms underlying the NHS defects.

Pericentric inversion in chromosome 1 and male infertility

Pericentric inversion in chromosome 1 was thought to cause male infertility through spermatogenic impairment, regardless of the breakpoint position. However, carriers of pericentric inversion in chromosome 1 have been reported with normal fertility and familial transmission. Here, we report two cases of pericentric inversion in chromosome 1. One case was detected in utero via amniocentesis, and the other case was detected after the wife of the carrier experienced two spontaneous abortions within 5 years of marriage. Here, the effect of the breakpoint position of the inversion in chromosome 1 on male infertility is examined and compared with the published cases. The association between the breakpoint of pericentric inversion in chromosome 1 and spermatogenesis is also discussed. Overall, the results suggest that the breakpoint position deserves attention from physicians in genetic counseling as inversion carriers can produce offspring.

MLL Gene Rearrangements in 174 Infants with Acute Leukemia.

Abstract 2537 Background. MLL gene rearrangements are the most common genetic events in infant leukemia. Up to date more than 100 various MLL rearrangements were described. Purpose. To evaluate the distribution of MLL rearrangements among infants (aged from 1 to 365 days) with both acute lymphoblastic leukemia (ALL) and acute myeloid leukemia (AML). Methods. 174 infants (117 ALL and 57 AML cases) were included in the current study. 11q23/MLL rearrangements were detected by chromosome banding analysis (CBA), fluorescence in-situ hybridization (FISH) and reverse-transcriptase PCR (RT-PCR). CBA was done according to standard procedure. FISH analysis using LSI MLL Dual Color, Break Apart Rearrangement Probe (Abbott Molecular, USA) was performed on at least 200 interphase nuclei and on all available metaphases. RT-PCR was performed as previously described (A. Borkhardt et al.,1994, N. Palisgaard et al., 1998, J. van Dongen et al., 1999). In 39 cases genomic DNA breakpoint was detected in MLL and translocation partner genes by long-distance inverse PCR (LDI-PCR). Exon-intron numbering of MLL gene was done according to I. Nilson et al, 1996. Results. 11q23/MLL rearrangements were revealed in 74 ALL patients (63.2%). Among this group MLL-AF4 was detected in the majority of cases (53.5%), less frequently were found MLL-MLLT1, MLL-MLLT3, MLL-MLLT10 and others (fig. 1a). Children with ALL under 6 months of age had significantly higher incidence of MLL rearrangements in comparison with older infants (84.0% vs. 47.8%, p<0.001). MLL-positive patients more frequently had BI-ALL and less frequently BII-ALL than infants without these rearrangements (p<0.001 for both). Fusion gene transcripts were sequenced in 26 MLL-rearranged ALL cases. Depending on breakpoint position within MLL and partner genes we detected 7 different types of MLL-AF4 fusion gene transcript, 3 types of MLL-MLLT1, 2 types of MLL-EPS15. The most common fusion site within MLL gene in ALL patients was exon 11, detected in 14 cases (53.8%). It was confirmed by LDI-PCR, that in addition to common breakpoint location in MLL gene (18 out of 27 cases in intron 11, 4 cases in intron 9) allowed to reveal less frequent breakpoint sites, like intron 12 (1 case), intron 10 (3 cases) and intron 7 (1 case). Interestingly, in the last case where LDI-PCR showed presence of MLL-AF4, this fusion gene transcript was not initially found by RT-PCR, because applied primer set did not cover exon 7. Moreover, due to lack of metaphases this patient was primary misclassified as MLL-rearranged, but MLL-AF4-negative. MLL rearrangements were found in 28 AML cases (49.1%). In AML patients the most common MLL rearrangements were MLL-MLLT10 (32% of cases) and MLL-MLLT3 (28%). Other ones were detected less frequently (fig. 1b). In AML patients frequency of MLL rearrangements was similar in children younger and older than 6 months (p=0.904). Among MLL-positive cases AML M5 were detected significantly more often and AML M7 significantly less frequent than in MLL-negative patients (p=0.024 and p=0.001, correspondingly). The most common breakpoint location within MLL gene in AML patients was intron 9, detected in 6 out of 12 cases (50%). Additional chromosomal abnormalities were revealed in 7 out of 21 MLL-positive AML patients with known karyotype (33%), while complex karyotype was detected in 5 cases (24%). Application of LDI-PCR allowed to verify rare MLL rearrangements, including MLL-AFF3 (1 ALL case), MLL-MYO1F (2 AML cases), MLL-SEPT6 (1 AML case), MLL-SEPT9 (1 AML case) In 4 ALL and 3 AML patients MLL rearrangements with concurrent 3'-deletion of MLL gene were found. 3'-deletion of MLL was not associated with breakpoint position in MLL gene and type of translocation partner gene. None of the patients with 3'-deletions had reciprocal fusion gene. Based on LDI-PCR data we assessed several mechanisms of fusion gene formation. Reciprocal translocations were detected in 29 cases, 3-way translocations in 3 cases, inversions in 5 cases, combination of inversion and insertion in 2 cases. Conclusion. In the current study we precisely characterized large cohort of MLL-rearranged infant acute leukemia patients. Combination of all available techniques, including cytogenetics, FISH, RT-PCR and LDI-PCR can lead to detailed verification of every single MLL rearrangement. Disclosures: No relevant conflicts of interest to declare.

Physical mapping of a 7A.7D translocation in the wheat–Thinopyrum ponticum partial amphiploid BE-1 using multicolour genomic in situ hybridization and microsatellite marker analysis

The absence of chromosome 7D in the wheat–Thinopyrum ponticum partial amphiploid BE-1 was detected previously by multicolour genomic in situ hybridization, sequential FISH (fluorescence in situ hybridization) using repetitive DNA probes, and SSR marker analysis. In the present study the previous cytogenetic and SSR marker analyses were expanded to include 25 other SSR markers assigned to wheat chromosomes 7A and 7D to confirm the presence of a 7A.7D translocation and to specify its composition. An almost complete chromosome 7A and a short chromosome segment derived from the terminal region of 7DL were detected, confirming the presence of a terminal translocation involving the distal regions of 7AL and 7DL. In both cases the position of the translocation breakpoint was different from that of known deletion lines. The identification of the 7AL.7DL translocation and its breakpoint position provides a new physical landmark for future physical mapping studies, opening up the possibility of more precise localization of genes or molecular markers within the terminal regions of 7DL and 7AL.

Identification and distinction among segmental duplication-deficiencies by fluorescence in situ hybridization (FISH)-adorned multivalent analysis

Most simple reciprocal translocation homozygotes and heterozygotes are euploid, and normal in genotype. However, translocation heterozygotes form six types of numerically balanced meiotic products. The cross of a translocation heterozygote with a normal individual can yield normal progeny, translocation heterozygotes, or any of four segmentally aneuploid duplication-deficient types (dp-dfs). Using metaphase I configuration analysis, most dp-dfs can be distinguished easily from normal and heterozygous translocations. However, identification of the four dp-df types is often impossible unless there is an appreciable karyotypic difference in arm size, relative breakpoint position, or a diagnostic cytological marker. Here we demonstrated the utility and facility of dp-df identification by means of meiotic fluorescence in situ hybridization (FISH) to adorn one chromosome arm with a molecular marker. The rationale is presented diagrammatically, and exemplified by identifying both adjacent-1 and adjacent-2 dp-dfs in Gossypium hirsutum. Polymorphism is not required among marker loci, so analysis of dp-dfs can proceed without requirement of sexual hybridization or sophisticated high-polymorphism marker systems. Besides facilitating the identification of dp-dfs, such an analysis can provide facile means to assign marker loci to chromosomes, arms, and segments. Integrative mapping of chromosomal, physical, and recombination maps will thus be facilitated. An ability to readily distinguish adjacent-1 and adjacent-2 types of dp-dfs should also enhance strategic derivation of other aneuploids, e.g., dp-df related monosomes and trisomes.Key words: Gossypium, cotton, duplication-deficiency, fluorescence in situ hybridization, repetitive DNA.