Am J Humi Genet 28:506-513, 1976
Hair Root Versus Red Cell Individual Phenotype in Sardinian Heterozygotes for G6PD Deficiency (Mediterranean Type) GIOVANNI ROMEO,1'3 ANTONIETTINA RINALDI,2 FRANCO URBANO, AND GIORGIo FILIPPI2 INTRODUCTION
Cell cloning has been used to identify heterozygotes for X-linked markers and to verify Lyon's hypothesis with respect to various X-linked loci [1-7]. In addition, mosaicism has been convincingly demonstrated with the hair follicles of heterozygotes for glucose-6-phosphate dehydrogenase (G6PD) and hypoxanthine guanine phosphoribosyltransferase (HGPRT) deficiencies [8-9]. These observations led Gartler et al. to conclude that hair roots originate from multiple cells belonging to one or more clonal areas of growth [10]. Variation among heterozygotes for an X-linked character may reflect the size of the primordial cell pool at the time of inactivation from which a tissue is developed, provided that variegation among different tissues can be proved and that cell selection does not play a discriminatory role in that tissue [ 11] or in the whole organism. Accordingly, the size of the primordial cell pool has been estimated to be eight cells for the erythropoietic cell lineage [11] and 10 cells or less for the scalp epidermis [8]. The present study confirms the work of Gartler et al. [8] which showed that variation among heterozygotes for G6PD deficiency can generate extreme phenotypic expression, simulating hair follicle phenotypes of homozygotes. MATERIALS AND METHODS
In the course of an extensive study on the genetics of G6PD performed in Sardinia [12], we analyzed the scalp hair follicles of 19 G6PD deficient and 19 control males, 4 G6PD deficient homozygous and 4 control females, and 20 heterozygotes. The criteria used for the ascertainment of G6PD deficient individuals and heterozygotes have been described in detail elsewhere [12]. Of the 20 heterozygotes, 16 were chosen at random Received February 13, 1976; revised May 10, 1976. This work was supported by grants from the Italian National Research Council. 1 Institute of Genetics, via Mezzocannone 8, University of Napoli and International Institute of Genetics and Biophysics, Napoli, Italy. 2 Institute of Genetics, University of Cagliari, Cagliari, Italy. A. Rinaldi was a Visiting Investigator at the Sloan-Kettering Institute for Cancer Research in New York City during 1975. 3 On leave from the University of Naples. Present address: Department of Genetics, Stanford University School of Medicine, Stanford, California 94305. © 1976 by the American Society of Human Genetics. All rights reserved.
506
PHENOTYPE VARIATION AND G6PD DEFICIENCY
507
from 77 individuals analyzed for blood phenotypes [12] and four because they exhibited more than 90% of normal red cells. Scalp hair roots were collected from different parts of the occipital region [8]. An average sample consisted of 15-30 hair roots with visible bulbs and sheaths (cut just above the sheath) which were placed on filter paper dampened in buffer A (Tris 0.1 M, pH 8.1, containing 0.001 M MgCl and 0.5 mM NADP) and stored in a freezer until analyzed. Each follicle was homogenized with a small glass Dounce homogenizer in 4.0 ml of buffer A; the homogenate was divided in three aliquots of 1.2 ml each: An aliquot of 25 il of 0.1 M NAD was added to two cuvettes, and these samples were kept at room temperature until analyzed. The reactions for G6PD, lactate dehydrogenase (LDH), and malate dehydrogenase (MDH) were started by adding the respective substrate (20 Al TABLE 1 DISTRIBUTION OF G6PD/LDH OF 755 SINGLE HAIR FOLLICLES FROM 66 SARDINIANS
(MALES AND FEMALES) HETERONORMAL G6PD PHENOTYPES
G6PD DEFICIENT PHENOTYPES
G6PD/LDH
M (19)
F (4)
0000.05 .................
17 113 50 5
1 12 6
18 125 56
3
8 1
0.05-0.10.................
0.10-0.15
.................
0.15-0.20 ................. 0.20-0.25 ................. 1 . 0.25-0.30 ...... 0.30-0.35 . ...... 0.35-0.40 . ...... 0.40-0.45 ................. ... 0.45-0.50 . . ..... . 0.50-0.55 ...... 0.55-0.60 ............ . ... 0.60-0.65 . ...... 0.65-0.70 . ...... 0.70-0.75 . ...... 0.75-0.80 ............ . ... 0.80-0.85 . ......
M+F (23) M (19)
... ...
...
...
...
...
...
15
...
... ...
...
14 24 10 10 12 5 9 3 1
...
4
...
...............
...
... ... ... ... ... ...
......
...
...
...
...
.
.
.
1.15-1.20
.................
Totals
...
...
...
2 1
...
...
...
...
...
......
1.05-1.10 ................... 1.10-1.15 .................
...
...
... .
...
....
... ...
...
...
6 5
...
3 3 3 3 ...
7 5 ...
2 1 ...
...
9 9
26 33 21 17 27 13 10 19 10 9 5 2 4
7
59 56 32 24 28 34 18
13 8 11 8 5 7 5 8 2 1 ...
2 3
...
...
... ...
... ...
...
...
2 ...
...
... ... ...
...
... ... ... ...
M+F (23) F (20)
...
...
...
...
............
...
...
...
0.95-1.00 1.00-1.05
...
... ...
...
...
0.90-0.95
...
9 9 23 30
...
...
0.85-0.90..
F (4)
ZYGOTE
...
2
186
22
208
181
38
219
328
var
.087 .001
.089 .001
SD SE
.002
.114 .003 .053 .011
.495 .032 .180 .013
.587 .035 .186 .030
.511 .034 .184 .012
.275 .043 .207 .011
x
...............
.034
.035 .003
NOTE.-First column indicates the lower and upper limits of each distribution arbitrarily chosen at intervals of Remaining columns indicate no. of hair follicles falling in each class. No. in parentheses = individuals examined.YG6PD (-) F -Y G6PD (-) M =.027 +4.011, t = 2.45, P
Hair Root Versus Red Cell Individual Phenotype in Sardinian Heterozygotes for G6PD Deficiency (Mediterranean Type) GIOVANNI ROMEO,1'3 ANTONIETTINA RINALDI,2 FRANCO URBANO, AND GIORGIo FILIPPI2 INTRODUCTION
Cell cloning has been used to identify heterozygotes for X-linked markers and to verify Lyon's hypothesis with respect to various X-linked loci [1-7]. In addition, mosaicism has been convincingly demonstrated with the hair follicles of heterozygotes for glucose-6-phosphate dehydrogenase (G6PD) and hypoxanthine guanine phosphoribosyltransferase (HGPRT) deficiencies [8-9]. These observations led Gartler et al. to conclude that hair roots originate from multiple cells belonging to one or more clonal areas of growth [10]. Variation among heterozygotes for an X-linked character may reflect the size of the primordial cell pool at the time of inactivation from which a tissue is developed, provided that variegation among different tissues can be proved and that cell selection does not play a discriminatory role in that tissue [ 11] or in the whole organism. Accordingly, the size of the primordial cell pool has been estimated to be eight cells for the erythropoietic cell lineage [11] and 10 cells or less for the scalp epidermis [8]. The present study confirms the work of Gartler et al. [8] which showed that variation among heterozygotes for G6PD deficiency can generate extreme phenotypic expression, simulating hair follicle phenotypes of homozygotes. MATERIALS AND METHODS
In the course of an extensive study on the genetics of G6PD performed in Sardinia [12], we analyzed the scalp hair follicles of 19 G6PD deficient and 19 control males, 4 G6PD deficient homozygous and 4 control females, and 20 heterozygotes. The criteria used for the ascertainment of G6PD deficient individuals and heterozygotes have been described in detail elsewhere [12]. Of the 20 heterozygotes, 16 were chosen at random Received February 13, 1976; revised May 10, 1976. This work was supported by grants from the Italian National Research Council. 1 Institute of Genetics, via Mezzocannone 8, University of Napoli and International Institute of Genetics and Biophysics, Napoli, Italy. 2 Institute of Genetics, University of Cagliari, Cagliari, Italy. A. Rinaldi was a Visiting Investigator at the Sloan-Kettering Institute for Cancer Research in New York City during 1975. 3 On leave from the University of Naples. Present address: Department of Genetics, Stanford University School of Medicine, Stanford, California 94305. © 1976 by the American Society of Human Genetics. All rights reserved.
506
PHENOTYPE VARIATION AND G6PD DEFICIENCY
507
from 77 individuals analyzed for blood phenotypes [12] and four because they exhibited more than 90% of normal red cells. Scalp hair roots were collected from different parts of the occipital region [8]. An average sample consisted of 15-30 hair roots with visible bulbs and sheaths (cut just above the sheath) which were placed on filter paper dampened in buffer A (Tris 0.1 M, pH 8.1, containing 0.001 M MgCl and 0.5 mM NADP) and stored in a freezer until analyzed. Each follicle was homogenized with a small glass Dounce homogenizer in 4.0 ml of buffer A; the homogenate was divided in three aliquots of 1.2 ml each: An aliquot of 25 il of 0.1 M NAD was added to two cuvettes, and these samples were kept at room temperature until analyzed. The reactions for G6PD, lactate dehydrogenase (LDH), and malate dehydrogenase (MDH) were started by adding the respective substrate (20 Al TABLE 1 DISTRIBUTION OF G6PD/LDH OF 755 SINGLE HAIR FOLLICLES FROM 66 SARDINIANS
(MALES AND FEMALES) HETERONORMAL G6PD PHENOTYPES
G6PD DEFICIENT PHENOTYPES
G6PD/LDH
M (19)
F (4)
0000.05 .................
17 113 50 5
1 12 6
18 125 56
3
8 1
0.05-0.10.................
0.10-0.15
.................
0.15-0.20 ................. 0.20-0.25 ................. 1 . 0.25-0.30 ...... 0.30-0.35 . ...... 0.35-0.40 . ...... 0.40-0.45 ................. ... 0.45-0.50 . . ..... . 0.50-0.55 ...... 0.55-0.60 ............ . ... 0.60-0.65 . ...... 0.65-0.70 . ...... 0.70-0.75 . ...... 0.75-0.80 ............ . ... 0.80-0.85 . ......
M+F (23) M (19)
... ...
...
...
...
...
...
15
...
... ...
...
14 24 10 10 12 5 9 3 1
...
4
...
...............
...
... ... ... ... ... ...
......
...
...
...
...
.
.
.
1.15-1.20
.................
Totals
...
...
...
2 1
...
...
...
...
...
......
1.05-1.10 ................... 1.10-1.15 .................
...
...
... .
...
....
... ...
...
...
6 5
...
3 3 3 3 ...
7 5 ...
2 1 ...
...
9 9
26 33 21 17 27 13 10 19 10 9 5 2 4
7
59 56 32 24 28 34 18
13 8 11 8 5 7 5 8 2 1 ...
2 3
...
...
... ...
... ...
...
...
2 ...
...
... ... ...
...
... ... ... ...
M+F (23) F (20)
...
...
...
...
............
...
...
...
0.95-1.00 1.00-1.05
...
... ...
...
...
0.90-0.95
...
9 9 23 30
...
...
0.85-0.90..
F (4)
ZYGOTE
...
2
186
22
208
181
38
219
328
var
.087 .001
.089 .001
SD SE
.002
.114 .003 .053 .011
.495 .032 .180 .013
.587 .035 .186 .030
.511 .034 .184 .012
.275 .043 .207 .011
x
...............
.034
.035 .003
NOTE.-First column indicates the lower and upper limits of each distribution arbitrarily chosen at intervals of Remaining columns indicate no. of hair follicles falling in each class. No. in parentheses = individuals examined.YG6PD (-) F -Y G6PD (-) M =.027 +4.011, t = 2.45, P
