WILLIAM D. LOUGHMAN, PhD, ABMG
393 Gravatt Dr., Berkeley, CA 94705
voice: (510) 841-7428   Internet: wdlkhl@attglobal.net


Cover: original report


CYTOGENETICS AND FANCONI'S ANEMIA:
EXPERIMENTAL AND OTHER STUDIES OF A FAMILY

DONNER LABORATORY

William D. Loughman
Ph.D. Thesis

May 1973

Prepared for the U.S. Atomic Energy Commision


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SUMMARY
[Pp. 187 - 191, transcribed]

          The genetic and cytogenetic literature concerning Fanconi's Anemia (FA) is reviewed. A case is made against the prevailing simplistic view of the etiology of FA. A rationale for further study is given.

          An unreported case of FA is presented. Physical and clinical descriptions are given for the FA case, her normal sib, both normal parents, and three deceased FA sibs.

          A pedigree of the kindred is given, containing 188 individuals in 7 generations. There is no parental consanguinity. The incidence of neoplasia and diabetes in the kindred is not elevated, but ascertainment is incomplete. Diabetes is present in the propositus's immediate antecedents. Infant mortality is elevated, and is 21% of live births in the paternal kindred.

          Marrow cells of the propositus contained no chromosome abnormalities during the 5 year period of observation. A small proportion of marrow cells contained numerous micronuclei.

          Cultured FA lymphocytes contained increased chromatid exchanges, gaps, and breaks, with multicentric chromosomes; there were virtually no other abnormalities. Chromatid exchanges were more common initially than chromatid breaks; this ratio reversed by the end of the study period. The frequency of chromatid exchanges declined exponentially through the course of treatment, with a half-time similar to the half-life of lymphocytes. Multicentric chromosomes were always less frequent than exchanges; they also declined with time. Chromatid breaks did not decline during the course of the disease. Endoreduplication was not common throughout the 5 year study, but was elevated following termination of treatment.

          The frequency of chromosome aberrations did not decline in the cultured FA lymphocytes with time in culture. This was interpreted as de novo creation of abnormalities in each cell generation.

          Homologous chromosomes were involved frequently in chromatid interchanges, but non-homologous exchange association was common. Long chromosomes were more common than short in chromatid exchanges. The increase above expectation may follow a power relationship.

          Mitotic index determinations on cultured FA lymphocytes revealed inconsistent and abnormal patterns, suggesting multiple cell populations or cell cycle abnormalities.

          In vitro survival of FA lymphocytes is reduced in two phases, one prominent after 48 hours of culture. Some of the reduced survival shown by dye-exclusion is spurious, suggesting a defect in cell membrane integrity.

          Lymphocytes from FA and normal individuals were cultured in the presence of each other's plasma; no changes in frequencies of chromosome abnormalities were observed. The experiment repeated with "conditioned" medium produced a seven-fold increase of simple chromatid breaks in normal cells.

          Electron micrographs of fresh FA lymphocytes were not different from those of normal individuals. Micrographs of cultured FA lymphocytes showed unusual lacunae in the endoplasmic reticulum and an accumulation of granules believed to be glycogen. Mitochondria were abnormally large, with reduced and unusually arranged cristae; ribosomes were reduced.

          Cell cycle analysis was performed on lymphocytes from all family members. The mother's G1 phase probably was prolonged; the durations of either or both of S and G1 were variable in both parents' cells. The normal sib had normal cell cycle parameters. The FA case's cell cycle times could not be determined. A large fraction of her dividing cells failed to label with radiothymidine; a much smaller fraction of both parents' cells also failed to label. The mother's cells showed some labelling late in G2 . Both the FA case and her mother, but not her father or normal sib, had a proportion of cells which labelled intensely. Additional evidence for a membrane defect in FA cells was obtained. Two populations of labelling FA cells were postulated; one would have an abnormal cell cycle.

          Failure of cultured lymphocytes to label with radiothymidine was offered as a clinically useful test for detection of the FA carrier.

          Both hexokinase and thymidine kinase activities were normal or elevated in cells of the FA case and her immediate family.

          The G1 chromosome repair time of the FA case was normal on the average, but the experimental results suggested either multiple cell populations or great variability of the repair time. Repair times determined for the other family member were the same as the controls'.

          The rate of chromosome-type aberration induction by x-rays during the first G1 in cultured FA lymphocytes was not elevated above control values. The rate of chromatid-type aberration-induction in the last G2 in FA lymphocytes was not elevated above control values. Determinations performed on family members' cells showed no difference from control values.

          On two occasions, almost three years apart, no significant aberrations were found in the chromosomes of cultured lymphocytes from the FA case's parents and normal sib.

          The cytogenetic abnormalities in cultured FA lymphocytes occur in a relatively simple pattern which is not consistent with the hypothesis of chromosome fragility and subsequent mis-repair. The "exchange" hypothesis of chromosome aberrations is more in accord with the bulk of the observational data given in this report.

          It is shown that the action on FA chromosomes of a physical agent is inconsistent with both observational and experimental evidence. The action of a virus, especially as the initiating agent in the exchange hypothesis, is consistent with but not proven by the data provided.

          The recessive gene hypothesis of FA causation was questioned. A more complex genetic basis for FA was considered likely.

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