Linkage Studies on X-Linked Ichthyosis in Sardinia
GIORGIO FILIPPI' AND P. MEERA KHAN2
The locus for X-linked ichthyosis has been shown as being a measurable distance
from the Xg locus (Kerr et al., 1964; Adam et al., 1966; Wells et al., 1966). The esti-
mate of recombination fraction between these two loci is 0.17, with 90% confidence
limits of 0.11 and 0.28 (Wells et al., 1966).
Among the families reported by Adam et al. (1966), there was one segregating also
for color blindness of the protan type with a ratio of two nonrecombinants (n.r.) to
four recombinants (r.), thus suggesting that the locus for X-linked ichthyosis is nearer
to the Xg locus than to the cluster of loci including those for color blindness, G6PD
deficiency, and hemophilia A.
The present report refers to similar investigations performed in Sardinia mainly
for the purpose of finding families where both X-linked ichthyosis and G6PD segre-
gate. This was expected to be a rather common finding among the propositi affected
by X-linked ichthyosis, since the frequency of G6PD deficiency in the lowland part
of this island was known to be of the order of + 30% (Siniscalco et al., 1966).
MATERIAL AND METHODS
Ascertainment of patients with X-linked ichthyosis. The criteria used for selection of
the cases to be studied were those recommended by Wells and Kerr (1966) and Wells
and Jennings (1967) as well as the obvious agreement with the pattern of X-linked
inheritance. In this way, of 17 suggestive cases ascertained through the files of the
Dermatologic Clinics of the Universities of Cagliari and Sassari, eight were found to
fulfill the criteria for a linkage study and were further investigated with their relatives
for G6PD variations, both quantitative and electrophoretic, color vision and Xg
groups.
Although only in two of these eight families (Pedigrees 3 and 8) could the mothers
of the propositi be proved to be heterozygous for X-linked ichthyosis in view of the
presence of the disease in other branches of the pedigree, all cases reported here are
considered to be segregational. This is because, in contrast with hemophilia, muscular
dystrophy, and some other X-linked conditions (Filippi and Macciotta, 1967), the
event of a fresh mutation can here be considered extremely improbable, for ichthyosis
Received April 1, 1968.
This work has been supported by a grant from the Italian National Research Council allocated to
the Department of Genetics, Faculty of Science, Rome; and by U.S. Public Health Service research
grant GM 13415 from the National Institutes of Health, allocated to the Department of Genetics,
University of Leiden, The Netherlands.
I Department of Genetics, Faculty of Science, University of Rome, Italy.
2 Department of Human Genetics, Faculty of Medicine, University of Leiden, The Netherlands.
564
X-LINKEI) ICHTHYOSIS 565
is: (1) relatively frequent, (2) of no serious handicap to its carriers, and (3) very likely
to escape detection.
G6PD tests. The classification of the G6PD phenotypes was performed with the
following methods:
1. Dye decoloration test of M\Iotulsky and Caampbell-Kraut (1961) (only for males).
2. The quantitative measurement of G6PD activity (both for males and females)
expressed in terms of hemoglobin and red cell count to overcome the disturbing effect
of thalassemia trait known to be of common occurrence on the island of Sardinia
(Siniscalco et al., 1966).
3. Localization of G6PD activity at the red cell level with the methemoglobin
elution test of Gall et al. (1965), which affords a reliable classification of the three
G(6PD phenotypes in females.
4. Electrophoresis of G6PD on cellogel (Rattazzi et al., 1967). All examples of
(G6PD deficiency were of the "i\Iediterranean" type.
Color vision test. The presence of color blindness was searched for by means of the
Ishihara tables.
Xg tests. The Xg typing was kindly performed by Dr. Patricia Tippett and Miss
June Gavin at the M.R.C. Blood Group Research Unit, London.
Lod scores. These were calculated as instructed by Maynard-Smith et al. (1961).
No a priori correction (Renwick and Schulze, 1964) was applied. Corrections for
ascertainment (el-values) were not used when G6PD was the test character, and the
heterozygosity at this locus was established by direct testing.
.4dditional studies. The presence of thalassemia was ascertained by means of the
following tests: red cell fragility, determination of 1\ICV, MCH, MVCHC, and per-
centage of Hb A2.
Since the phase of the maternal genotype was deduced from the examination of
the maternal grandparents, it was felt necessary to exclude illegal paternity by study-
ing the segregation of the following autosomal markers: ABO, 1\INS, Rh, Kell, P, Lu-
theran, and Duffy blood groups; and serum haptoglobins. Adequate amounts of sera
and red cells from each individual were stored under liquid nitrogen for future needs.
RESULTS
The pedigrees of the eight propositi with X-linked ichthyosis are given in Figure 1.
None was found to segregate for color blindness. No case of illegal paternity was
found. One pedigree (No. 1) was found to give information only for the linkage ich-
thyosis-G6PD, one (No. 6) only for ichthyosis-Xg, and two (No. 2 and No. 8) for
both linkage comparisons. Pedigree No. 5 is likely to yield much more data on the
linkage ichthyosis-G6PD, should the four daughters, heterozygous at the G6PD
locus, eventually have male offspring with X-linked ichthyosis.
Additional information on these families is stored in the archives of the Depart-
ment of Genetics, Faculty of Science, University of Rome.
.,I nalysis of the Informative Pedigrees
Pedigree No. 1. Although the mother of the propositus is dead, it is clear that she
must have been doubly heterozygous in repulsion at loci for X-linked ichthyosis and
566 FILIPPI AND KHAN
G6PD deficiency. This conclusion is based on the finding that her youngest daughter
shows no G6PD-deficient red cells with the methemoglobin-elution test, and therefore
she must be considered homozygous for the normal G6PD allele. Gall et al. (1965)
have shown that their method allows a reliable diagnosis even in the heterozygotes
for the mildly deficient G6PD allele which occurs among American Negroes. In our
experience, the chance of misclassification of a heterozygote for the G6PD-deficient
Mediterranean allele is practically zero when the G6PD activity is studied at the cell
level, since we found that among several genetically proved heterozygous females
PEDIGREE 1 PEDIGREE 2 PEDIGREE 3
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ICHTHYOSIS: Affected * absence of sign stands for not affected
)" propositus(for ichthyosis), tdeceased
FIG. 1. Pedigrees of eight families in which X-linked ichthyosis is segregating
X-LINKED ICHTHYOSIS 56/7
none had a proportion of deficient red cells below 8-10cc, the great majority falling
within the range 20-80%/c (1\I. Siniscalco et al., data unpublished).
In conclusion, the score for the linkage ichthyosis-(16PD in this pedigree is 2 n.r.:
3 r., with phase known.
Pedigree No. 2. This pedigree scores for both linkage comparisons, but with phase
unknown: ichthyosis-G6PD, z1 = 3:0; ichthyosis-Xg, zi = 3:0, el = 2:1.
Pedigree No. 6. This pedigree scores only for ichthyosis-Xg, with phases unknown:
zt = 3: 0, el = 2: 1. The presence of G6PD deficiency in this family may be excluded,
since the mother of the propositus has no deficient red cells by the methemoglobin-
elution test.
Pedigree AXo. 8. In Generation 11, sibs II-3 and 11-7 score for ichthyosis-G6PD:
z, = 1: 1, e1 = 2:0. The possibility that their mother (1-2) might have been homozy-
gous for X-linked ichthyosis is excluded on the basis of family history, absence of
consanguinity, and the low incidence of the mutant in question.
Generation III: Sibs 111-8 and 111-9 score for ichthyosis-G6PD phase unknown:
z, = 1:1. The presence of G6PD deficiency and ichthyosis in the dead child (111-9)
is indirectly but unequivocally established since (1) the boy suffered several attacks
of clinical favism and is reported to have died at age five of this disease, and (2) the
mother reports with certainty that the child had the same skin troubles as her two
brothers.
Generation IV: Sibs IV-2, IV-3, IV-5, and IV-6 score for ichthyosis-G6PD phase
known; 3 n.r.: 1 r. Sibs IV-7 and IV-8 score for ichthyosis-Xg, phase known,
1 n.r.:1 r.
DISCUSSION
Table 1 gives the summary of the lod scores for the two linkage comparisons ich-
thyosis-G6PD and ichthyosis-Xg. Owing to the scarcity of data, it is not worthwhile
to calculate the antilogs and to draw the probability curves for a precise estimate of
the recombination fractions. It is obvious, however, that the present data strongly
support the hypothesis that the locus for X-linked ichthyosis is at nonmeasurable
linkage distance from the G6PD locus and add to the evidence that ichthyosis is
within measurable distance of the Xg locus.
It is hoped that in the near future the families reported in Table 1 may be typed for
the Xm system and that in the coming years many more scorable sibs may become
available from the present material. This was the main reason we were encouraged to
publish the present report, even though the data are by themselves not sufficient
to draw a final conclusion on the relative distances among the three above-mentioned
loci.
SUMMARY
Eight Sardinian families segregating for X-linked ichthyosis were studied for G6PD
deficiency, color vision, and the Xg groups. Four of them gave information for the
linkages ichthyosis-G6PD and ichthyosis-Xg. The data suggest that the locus for
X-linked ichthyosis is at a nonmeasurable distance from the G6PD locus. The data
support measurable linkage between the loci for ichthyosis and the Xg blood groups.
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X-LINKED ICHTHYOSIS 569
ACKNOWLEDGMENTS
We express our deep gratitude to Professor Pinetti, Director of Dermatologic Clinic,
University of Cagliari, and to Professor Rebuffo, Director of Dermatologic Clinic, University
of Sassari, for referring the majority of these cases; to Dr. R. R. Race and Dr. Ruth Sanger,
M.R.C. Blood Group Research Unit, Lister Institute, London, for the Xg typing; to Professor
E. Van Loghem and Dr. L. E. Nijenhuis, Central Laboratory of the Nederlands, Red Cross
Blood Transfusion Service, Amsterdam, for typing of other blood groups; to Dr. L. V. Went
and Miss G. Tavernier for the cellular localization of G6PD activity; and to Professor M.
Siniscalco for his inspiring advice and help throughout the entire investigation.
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