Study of complex structural variations of X-linked deafness-2 (DFNX2) based on single-molecule sequencing

X-linked deafness-2 (DFNX2) is cochlear incomplete partition type III (IP-III), one of inner ear malformations characterized by an abnormally wide opening in the bone separating the basal turn of the cochlea from the internal auditory canal, fixation of the stapes and cerebrospinal fluid (CSF) gusher upon stapedectomy or cochleostomy. The causative gene of DFNX2 was POU3F4. To investigate the genetic causes of X-linked deafness-2 (DFNX2) and compare the efficiency of different sequencing methods, twelve unrelated patients were enrolled in this study. Targeted next-generation sequencing (NGS) and long-read sequencing were used to analyze the genetic etiology of DFNX2. Six variants of POU3F4 were identified in this cohort by NGS. Three patients with a negative diagnosis based on NGS were enrolled in further long-read sequencing. Two of them were all found to carry structural variations (SVs) on chromosome X, consisting of an 870-kb deletion (DEL) at upstream of POU3F4 and an 8-Mb inversion (INV). The 870-kb DEL may have been be due to non-homologous end joining, while non-allelic homologous recombination within a single chromatid may have accounted for the 8-Mb INV. Common POU3F4 mutations in DFNX2 included point mutations, small insertions and deletions (INDELs), and exon mutations, which can be detected by Sanger sequencing and NGS. Single-molecule long-read sequencing constitutes an additional and valuable method for accurate detection of pathogenic SVs in IP-III patients with negative NGS results.

Meiotic Alterations in CAG Repeat Tracts

We have investigated meiotic changes in CAG repeat tracts embedded in a yeast chromosome. Repeat tracts undergo either conversion events between homologs or expansion and contraction events that appear to be confined to a single chromatid. We did not find evidence for conversion of tract interruptions or excess exchange of flanking markers.

Merotelic Kinetochore Orientation Is a Major Mechanism of Aneuploidy in Mitotic Mammalian Tissue Cells

In mitotic cells, an error in chromosome segregation occurs when a chromosome is left near the spindle equator after anaphase onset (lagging chromosome). In PtK1 cells, we found 1.16% of untreated anaphase cells exhibiting lagging chromosomes at the spindle equator, and this percentage was enhanced to 17.55% after a mitotic block with 2 μM nocodazole. A lagging chromosome seen during anaphase in control or nocodazole-treated cells was found by confocal immunofluorescence microscopy to be a single chromatid with its kinetochore attached to kinetochore microtubule bundles extending toward opposite poles. This merotelic orientation was verified by electron microscopy. The single kinetochores of lagging chromosomes in anaphase were stretched laterally (1.2–5.6-fold) in the directions of their kinetochore microtubules, indicating that they were not able to achieve anaphase poleward movement because of pulling forces toward opposite poles. They also had inactivated mitotic spindle checkpoint activities since they did not label with either Mad2 or 3F3/2 antibodies. Thus, for mammalian cultured cells, kinetochore merotelic orientation is a major mechanism of aneuploidy not detected by the mitotic spindle checkpoint. The expanded and curved crescent morphology exhibited by kinetochores during nocodazole treatment may promote the high incidence of kinetochore merotelic orientation that occurs after nocodazole washout.

Random single chromatid and nonrandom double chromatid type segregation of human acrocentric chromosomes in BrdU-labeled mitoses

Chromatid segregation was analyzed using satellite association of 5-bromodeoxyuridine (BrdU) differentially stained acrocentric chromosomes of human leukocytes. Data were classified into cis and trans configurations in second and third division cycles. It was found that single chromatid types have random segregation (1:1) while nonrandom segregation was noted for double chromatid types. The nonrandom segregation hypothesis of earlier investigators needs to be reexamined.Key words: chromatid segregation, BrdU, mitosis, acrocentric.

Chromosome behavior after laser microirradiation of a single kinetochore in mitotic PtK2 cells.

The role of the kinetochore in chromosome movement was studied by 532-nm wavelength laser microirradiation of mitotic PtK2 cells. When the kinetochore of a single chromatid is irradiated at mitotic prometaphase or metaphase, the whole chromosome moves towards the pole to which the unirradiated kinetochore is oriented, while the remaining chromosomes congregate on the metaphase plate. The chromatids of the irradiated chromosome remain attached to one another until anaphase, at which time they separate by a distance of 1 or 2 micrometers and remain parallel to each other, not undergoing any poleward separation. Electron microscopy shows that irradiated chromatids exhibit either no recognizable kinetochore structure or a typical inactive kinetochore in which the tri-layer structure is present but has no microtubules associated with it. Graphical analysis of the movement of the irradiated chromosome shows that the chromosome moves to the pole rapidly with a velocity of approximately 3 micrometers/min. If the chromosome is close to one pole at irradiation, and the kinetochore oriented towards that pole is irradiated, the chromosome moves across the spindle to the opposite pole. The chromosome is slowed down as it traverses the equatorial region, but the velocity in both half-spindles is approximately the same as the anaphase velocity of a single chromatid. Thus a single kinetochore moves twice the normal mass of chromatin (two chromatids) at the same velocity with which it moves a single chromatid, showing that the velocity with which a kinetochore moves is independent, within limits, of the mass associated with it.

EFFECT OF TWO CHEMOSTERILANTS APHOLATE AND HEMPA ON THE BONE-MARROW CHROMOSOMES OF MICE

Mice were injected with 0.1% solution of apholate and hempa at the rate of 1.5 ml per 100 gm body weight and their bone-marrow cells were fixed at different intervals between 15 min and 144 hr. Specimens injected with distilled water served as controls.Only three aberrations were found in 4,100 metaphases in the control series. In the hempa-treated series, out of 4,100 metaphases examined one chromatid break, one subchromatid break, two gaps and one exchange were found at 24 hr only.In the apholate-treated series aberrations were absent only at 15 min and 144 hr post injection. At other times the aberrations were single chromatid breaks, subchromatid breaks, aniso- and isochromatid breaks, constrictions, gaps and lesions and exchanges. The aberration peak of 28.2% was reached at 24 hr, and fell to 0.3% at 120 hr. The breaks were non-randomly distributed between and within chromosome groups. Groups I and II and distal region of all affected groups were most susceptible.The mode of action of apholate and hempa in the induction of chromatid aberrations in mice, their localization and non-random distribution are discussed. The role of the aziridine groups in inducing aberrations has been considered. It is suggested that the breaks were due to some direct action and that the chromatids of mice contain some weaker regions.