- Email: [email protected]
Genetic selection and folate intake during pregnancy
RESEARCH LETTERS
9 8
E Muñoz-Moran, J L Dieguez-Lucena, N Fernandez-Arcas, S Peran-Mesa, A Reyes-Engel
Without loss of appetite (n=6) 4559 m 490 m
7 Serum leptin (ng/mL)
Genetic selection and folate intake during pregnancy
With loss of appetite (n=12) 4559 m 490 m
6 5 4 3 2 1 0 1
4
8
12
16
10 00 h
20
24
28
32
10 00 h
Time (h) Serum leptin concentrations of 18 volunteers at 490 m (Lausanne, Switzerland) and at 4559 m (Capanna Margherita, Switzerland) with and without loss of appetite An increase in integrated serum leptin levels (mean area under the curve) from 53·8 (SD 13·8) ng/ml per h to 66·3 (16·2) ng/ml per h was found in individuals with loss of appetite (1·20 hours, p=0·008), but not in volunteers without loss of appetite (38·7 [6·4] ng/mL per h [490 m] vs 40 8 [13·2] mg/mL per h [4559 m], p=0·35).
with AMS, compared with concentrations at 490 m (AMS was defined as a functional Lake Louise Score of more than 1 [n=10, p=0·028].5 Statistics were Mann-Whitney test and Wilcoxon tests. Effects due to change in plasma volume were excluded. Individuals with loss of appetite showed a tendency to higher leptin concentrations at baseline than those without (p=0·1, figure), but mean body mass indices were not significantly different between subgroups. In two independent studies, elevated leptin concentrations at high altitude were found to be associated with loss of appetite. Thus, leptin may be a player in the altered neuroendocrine regulation of energy homeostasis at high altitude, leading to loss of appetite, increased energy expenditure and weight loss.
The Ala225Val mutation (677C➝T) for the methylenetetrahydrofolate reductase (MTHFR) gene has been associated with a thermolabile enzyme with a decreased activity that can provide an increase in plasma homocysteine concentrations.1 Several reports have studied the relation of this polymorphism with cardiovascular diseases as well as schizophrenia, depression, and cancer. The frequency of the homozygous VV genotype, however, varies geographically in Europe from 6–10% in northern countries to 13–18% in the Mediterranean population. We have studied the evolution with age of the allele and genotype frequencies of this polymorphism in a healthy population from southern Spain (n=695) that was homogeneously distributed by age. We excluded people older than 40 years to prevent changes in frequency due to the possible implication of this gene in different pathologies and to nutritional habits. All the individuals were genotyped for the insertion/deletion polymorphism of the angiotensinconverting-enzyme gene, a locus different to the one studied, to know the genetic homogeneity of the adult and young populations. The allele and genotype frequencies did not differ significantly when both populations were compared. Unexpectedly, we found a substantial increase in frequency of the VV homozygous genotype in individuals younger than 20 years (figure). In a more detailed study, we found a shift in the VV genotype frequency, from 13% to 26%, that started in people born between 1977 and 1982 and that remained at this high proportion (figure). We found also that the population from which these individuals were derived was in Hardy-Weinberg equilibrium. In 1982 early folate treatment for all pregnant women was recommended by the Spanish national health service to Genotype
3 4
5
Neuroendocrine Unit (M Tschöp; e-mail [email protected]) and Mountain Medicine Study Group, Department of Medicine and Department of Clinical Pharmacology, Innenstadt University Hospital, 80336 Munich, Germany; Departement de Medecine, Centre Hospitalier Universitaire Vaudois, Lausanne, Switzerland; and Institute of Sports Medicine, Department of Medicine, Heidelberg, Germany
1120
VV
V
(n=327)
*
60
*
40
30 20
†
20
10 Frequency (%)
2
Sampson JB, Cymerman A, Burse RL, Maher JT, Rock PB. Procedures for the measurement of acute mountain sickness. Aviat Space Environ Med 1983; 54: 1063–73. Westerterp KR, Kayser B, Wouters L, Le-Trong JL, Richalet JP. Energy balance at high altitude of 6542 m. J Appl Physiol 1994; 77: 862–66. Auwerx J, Staels B. Leptin. Lancet 1998; 351: 737–42. Tschöp M, Wu Z, Bidlingmaier M, Heiman ML, Strasburger CJ. Generation of monoclonal antibodies and establishment of a sensitive sandwich immunoassay for the measurement of human serum leptin Exp Clin Endocrinol Diabetes 1998; 106 (Suppl.1): 40. The Lake Louise Consensus on the Definition and Quantification of Altitude Illness in: Sutton JR, Coates G, Houston CS, eds. Hypoxia and Mountain Medicine, Proceedings of the 7th International Hypoxia Symposium held at Lake Louise, Canada, February 1991: 327–30.
50
A
80
40
We thank Z Wu, M Bidlingmaier, H Hautmann, R Fischer, R Riepl, M Töpfer, K Morrison (Innenstadt University Hospital, Munich), M Heiman (Lilly Research Laboratories, Indianapolis) and M Appenzeller (Department de Medicine, CHUV, Lausanne) for their support and advice. 1
AV
(n=368)
Allele
AA
60
0
0 0–20
21–40
0–20
21–40
W genotype 30 (n=91) (n=87) (n=95) (n=95)
20 (n=85)
(n=80) (n=83) (n=79)
10 0 0–5
6–10 11–15 16–20 21–25 26–30 31–35 36–40 Age (years)
†=p<0·001
*=p<0·0001 (2 test) n=total number of studied individuals divided by age
Frequency of MTHFR 677C➝T mutation in southern Spain
THE LANCET • Vol 352 • October 3, 1998
RESEARCH LETTERS
prevent neural-tube defects.2 This treatment was started by physicians prescribing multivitamins and folic acid some years earlier; 3% of pregnant women received multivitamins in 1976, which increased to 10% in 1977, 35% in 1982, and 55% in 1986.3 An association has been reported between women with the hyperhomocysteinaemia and the A225V mutation and recurrent miscarriages and that these women have normal births after folic acid and pyridoxine supplementation.4 Therefore, a hypothesis can be made that there is an association between early folate supplementation during pregnancy and the increased number of babies born with the VV genotype, especially in VV mothers. The Mediterranean diet may also influence differences in genotype frequencies between northern and southern Europe. This study was supported by the Spanish Ministry of Education and Culture, Grants SAF95–0804 and SAF98–0163 1
2
3
4
Frosst P, Blom HJ, Goyette P, et al. A candidate genetic risk factor for cardiovascular disease: a common mutation in methylenetetrahydrofolate reductase. Nat Genet 1995; 10: 111–13. Smithells RW, Sheppard S, Schorah CJ, et al. Possible prevention of neural tube defects by periconceptional vitamin supplementation. Lancet 1980; i: 339–40. Salvador J, Martinez-Frias ML, Rodriguez-Pinilla E. Consumo de medicamentos por la mujer embarazada en España: perfil de una muestra de la población en los años 1976–1986. Madrid: Miniterio de Sanidad y Consumo, 1989. Quere I, Bellet H, Hoffet M, et al. A woman with five consecutive fetal deaths: case report and retrospective analysis of hyperhomocysteinemia prevalence in 100 consecutive women with recurrent miscarriages. Fertil Steril 1998; 69: 152–54.
Department of Biochemistry and Molecular Biology, Facultad de Medicina, Universidad de Málaga, 29071-Málaga, Spain, (A Reyes-Engel; e-mail [email protected]); and Laboratorio Endocrinologia Molecular, Hospital Regional Carlos Haya, Malaga
germ cell, ovarian follicle, or corpora albicans were detected in these areas. Left gonad showed occasional seminiferous tubule-like structures devoid of germ cells and spermatozoa. Southern hybridisation and FISH with a Y-chromosomespecific probe established the presence of Y chromosome in blood and gonadal tissues and ruled out the possibility of mosaicism. PCR amplification and sequencing of the PCR products of the SRY, SOX9, and ZFY genes showed no mutation. X-chromosome specific STR analysis revealed the paternal origin of the extra X chromosome. There are reports of mosaicism (46,XX/47,XXY and 46,XX/47XXY/48XXYY) leading to true hermaphroditism.5 But in the present case mosaicism was ruled out. Generally, individuals possessing 47,XXY karyotype have male phenotype. However, in the present case in spite of the presence of Y chromosome and the normal SRY, SOX9, and ZFY genes, the individual has a female phenotype. This strongly suggests that the phenotypic sex in this case might be due to the involvement of other sex determining genes. We thank K M Gun for referring the case. Financial support of the Department of Biotechnology, Government of India, for creating Automated DNA Sequencing facility and providing support for carrying out the work is gratefully acknowledged. We are grateful to A J Rachel for her help in doing fluorescence in-situ hybridisation. 1
2
3 4 5
A 47,XXY female
Klinefelter HF, Reifenstein EC, Albright F. Syndrome characterized by gynecomastia, aspermatogenesis without a-Leydigism, and increased excretion of follicle stimulating hormone. J Clin Endocrinol 1942; 2: 615–27. Sinclair AH, Berta P, Palmer MS, et al. A gene from the human sexdetermining region encodes a protein with homology to a conserved DNA-binding motif. Nature 1990; 346: 240–44. Pevny LH, Lovell-Bedge. SOX genes find their feet. Curr Opin Genet Develop 1997; 7: 338–44. Cameron FJ, Sinclair AH. Mutation in SRY and SOX9: testisdetermining genes. Hum Mutation 1997; 9: 388–95. Sano K, Terashima K, Tanaka Y, Sasaki Y. Four cases of true hermaphroditism. Hinyokika Kiyo 1995; 41: 73–77.
Centre for Cellular and Molecular Biology, Hyderabad 500 007, India (L Singh); Institute of Reproductive Medicine, Calcutta; Centre for DNA Fingerprinting and Diagnostics, Hyderabad
Kumarasamy Thangaraj, Nalini J Gupta, Baidyanath Chakravarty, Lalji Singh
Klinefelter syndrome (47,XXY) with male phenotype is the most common sex chromosomal abnormality.2 It is believed that SRY (sex determining region on Y) is the major gene necessary to induce the undifferentiated bipotential gonadal primordium to develop as testis.2 Recently, several SRY-boxrelated (SOX) genes have been identified on autosomes.3 Mutations in the SRY or SOX gene have been implicated in sex reversal.4 We report an individual having 47,XXY chromosome constitution, normal SRY, SOX9, and ZFY genes and yet with a female phenotype. A 15-year-old phenotypic female has well developed breasts; prominent labioscrotal folds; an enlarged clitoris; feminine pubic hair distribution; urethra in the centre of the vestibule; a urogenital sinus 5 cm deep; a uterus; prepubertal in shape and size, palpable per rectum; and a cervix which could not be palpated. Ultrasonography of the abdomen revealed normal shape of urinary bladder with smooth and regular walls. Uterus was small in size, with normal shape and outline. Myometrium was homologous, cavity was empty, midline echo was normal. Laparoscopic assessment revealed a normal uterus. Right ovary was normal in size and shape with a cyst and a normal Fallopian tube. Left ovary was not seen as it was hidden beneath an epithelial fold. Chromosome analysis showed 47,XXY karyotype in all 100 metaphases analysed. Hormone profiles showed average female range of corticotrophin, T3, T4, thyrotrophin, and testosterone. Progesterone and prolactin were higher when compared with normal female levels. Histology of the right gonad showed features compatible with ovarian stroma. No
THE LANCET • Vol 352 • October 3, 1998
Acute neuropathy after exposure to sun in a patient treated with St John’s Wort Geoffrey M Bove
Extracts or whole-herb preparations of St John’s Wort (Hypericum perforatum) are popular to treat depression. Photosensitivity is known to be a possible side effect. We report one patient who, while taking St John’s Wort, developed subacute polyneuropathy after exposure to sun. A 35-year-old woman took ground whole St John’s Wort (500 mg/day) for mild depression, after reading a magazine article. After 4 weeks, she developed stinging pain on her face and dorsum of both hands (areas exposed to the sun). Spontaneous pain was mild but worsened during and after being in the sun. Pain was provoked by minimal mechanical stimuli such as light touch or air movement. Cooling increased and warming decreased the pain. She sought help when the same symptoms developed on her arms and legs a few hours after sunbathing, and were limited to the exposed skin. She was examined 2 weeks after the pain started. There were no skin burns on examination. Light brushing, a gust of air at room temperature, and cold (~5–10OC) were acutely painful and outlasted the stimulus for seconds afterwards, consistent with allodynia. These symptoms were restricted to
1121
9 8
E Muñoz-Moran, J L Dieguez-Lucena, N Fernandez-Arcas, S Peran-Mesa, A Reyes-Engel
Without loss of appetite (n=6) 4559 m 490 m
7 Serum leptin (ng/mL)
Genetic selection and folate intake during pregnancy
With loss of appetite (n=12) 4559 m 490 m
6 5 4 3 2 1 0 1
4
8
12
16
10 00 h
20
24
28
32
10 00 h
Time (h) Serum leptin concentrations of 18 volunteers at 490 m (Lausanne, Switzerland) and at 4559 m (Capanna Margherita, Switzerland) with and without loss of appetite An increase in integrated serum leptin levels (mean area under the curve) from 53·8 (SD 13·8) ng/ml per h to 66·3 (16·2) ng/ml per h was found in individuals with loss of appetite (1·20 hours, p=0·008), but not in volunteers without loss of appetite (38·7 [6·4] ng/mL per h [490 m] vs 40 8 [13·2] mg/mL per h [4559 m], p=0·35).
with AMS, compared with concentrations at 490 m (AMS was defined as a functional Lake Louise Score of more than 1 [n=10, p=0·028].5 Statistics were Mann-Whitney test and Wilcoxon tests. Effects due to change in plasma volume were excluded. Individuals with loss of appetite showed a tendency to higher leptin concentrations at baseline than those without (p=0·1, figure), but mean body mass indices were not significantly different between subgroups. In two independent studies, elevated leptin concentrations at high altitude were found to be associated with loss of appetite. Thus, leptin may be a player in the altered neuroendocrine regulation of energy homeostasis at high altitude, leading to loss of appetite, increased energy expenditure and weight loss.
The Ala225Val mutation (677C➝T) for the methylenetetrahydrofolate reductase (MTHFR) gene has been associated with a thermolabile enzyme with a decreased activity that can provide an increase in plasma homocysteine concentrations.1 Several reports have studied the relation of this polymorphism with cardiovascular diseases as well as schizophrenia, depression, and cancer. The frequency of the homozygous VV genotype, however, varies geographically in Europe from 6–10% in northern countries to 13–18% in the Mediterranean population. We have studied the evolution with age of the allele and genotype frequencies of this polymorphism in a healthy population from southern Spain (n=695) that was homogeneously distributed by age. We excluded people older than 40 years to prevent changes in frequency due to the possible implication of this gene in different pathologies and to nutritional habits. All the individuals were genotyped for the insertion/deletion polymorphism of the angiotensinconverting-enzyme gene, a locus different to the one studied, to know the genetic homogeneity of the adult and young populations. The allele and genotype frequencies did not differ significantly when both populations were compared. Unexpectedly, we found a substantial increase in frequency of the VV homozygous genotype in individuals younger than 20 years (figure). In a more detailed study, we found a shift in the VV genotype frequency, from 13% to 26%, that started in people born between 1977 and 1982 and that remained at this high proportion (figure). We found also that the population from which these individuals were derived was in Hardy-Weinberg equilibrium. In 1982 early folate treatment for all pregnant women was recommended by the Spanish national health service to Genotype
3 4
5
Neuroendocrine Unit (M Tschöp; e-mail [email protected]) and Mountain Medicine Study Group, Department of Medicine and Department of Clinical Pharmacology, Innenstadt University Hospital, 80336 Munich, Germany; Departement de Medecine, Centre Hospitalier Universitaire Vaudois, Lausanne, Switzerland; and Institute of Sports Medicine, Department of Medicine, Heidelberg, Germany
1120
VV
V
(n=327)
*
60
*
40
30 20
†
20
10 Frequency (%)
2
Sampson JB, Cymerman A, Burse RL, Maher JT, Rock PB. Procedures for the measurement of acute mountain sickness. Aviat Space Environ Med 1983; 54: 1063–73. Westerterp KR, Kayser B, Wouters L, Le-Trong JL, Richalet JP. Energy balance at high altitude of 6542 m. J Appl Physiol 1994; 77: 862–66. Auwerx J, Staels B. Leptin. Lancet 1998; 351: 737–42. Tschöp M, Wu Z, Bidlingmaier M, Heiman ML, Strasburger CJ. Generation of monoclonal antibodies and establishment of a sensitive sandwich immunoassay for the measurement of human serum leptin Exp Clin Endocrinol Diabetes 1998; 106 (Suppl.1): 40. The Lake Louise Consensus on the Definition and Quantification of Altitude Illness in: Sutton JR, Coates G, Houston CS, eds. Hypoxia and Mountain Medicine, Proceedings of the 7th International Hypoxia Symposium held at Lake Louise, Canada, February 1991: 327–30.
50
A
80
40
We thank Z Wu, M Bidlingmaier, H Hautmann, R Fischer, R Riepl, M Töpfer, K Morrison (Innenstadt University Hospital, Munich), M Heiman (Lilly Research Laboratories, Indianapolis) and M Appenzeller (Department de Medicine, CHUV, Lausanne) for their support and advice. 1
AV
(n=368)
Allele
AA
60
0
0 0–20
21–40
0–20
21–40
W genotype 30 (n=91) (n=87) (n=95) (n=95)
20 (n=85)
(n=80) (n=83) (n=79)
10 0 0–5
6–10 11–15 16–20 21–25 26–30 31–35 36–40 Age (years)
†=p<0·001
*=p<0·0001 (2 test) n=total number of studied individuals divided by age
Frequency of MTHFR 677C➝T mutation in southern Spain
THE LANCET • Vol 352 • October 3, 1998
RESEARCH LETTERS
prevent neural-tube defects.2 This treatment was started by physicians prescribing multivitamins and folic acid some years earlier; 3% of pregnant women received multivitamins in 1976, which increased to 10% in 1977, 35% in 1982, and 55% in 1986.3 An association has been reported between women with the hyperhomocysteinaemia and the A225V mutation and recurrent miscarriages and that these women have normal births after folic acid and pyridoxine supplementation.4 Therefore, a hypothesis can be made that there is an association between early folate supplementation during pregnancy and the increased number of babies born with the VV genotype, especially in VV mothers. The Mediterranean diet may also influence differences in genotype frequencies between northern and southern Europe. This study was supported by the Spanish Ministry of Education and Culture, Grants SAF95–0804 and SAF98–0163 1
2
3
4
Frosst P, Blom HJ, Goyette P, et al. A candidate genetic risk factor for cardiovascular disease: a common mutation in methylenetetrahydrofolate reductase. Nat Genet 1995; 10: 111–13. Smithells RW, Sheppard S, Schorah CJ, et al. Possible prevention of neural tube defects by periconceptional vitamin supplementation. Lancet 1980; i: 339–40. Salvador J, Martinez-Frias ML, Rodriguez-Pinilla E. Consumo de medicamentos por la mujer embarazada en España: perfil de una muestra de la población en los años 1976–1986. Madrid: Miniterio de Sanidad y Consumo, 1989. Quere I, Bellet H, Hoffet M, et al. A woman with five consecutive fetal deaths: case report and retrospective analysis of hyperhomocysteinemia prevalence in 100 consecutive women with recurrent miscarriages. Fertil Steril 1998; 69: 152–54.
Department of Biochemistry and Molecular Biology, Facultad de Medicina, Universidad de Málaga, 29071-Málaga, Spain, (A Reyes-Engel; e-mail [email protected]); and Laboratorio Endocrinologia Molecular, Hospital Regional Carlos Haya, Malaga
germ cell, ovarian follicle, or corpora albicans were detected in these areas. Left gonad showed occasional seminiferous tubule-like structures devoid of germ cells and spermatozoa. Southern hybridisation and FISH with a Y-chromosomespecific probe established the presence of Y chromosome in blood and gonadal tissues and ruled out the possibility of mosaicism. PCR amplification and sequencing of the PCR products of the SRY, SOX9, and ZFY genes showed no mutation. X-chromosome specific STR analysis revealed the paternal origin of the extra X chromosome. There are reports of mosaicism (46,XX/47,XXY and 46,XX/47XXY/48XXYY) leading to true hermaphroditism.5 But in the present case mosaicism was ruled out. Generally, individuals possessing 47,XXY karyotype have male phenotype. However, in the present case in spite of the presence of Y chromosome and the normal SRY, SOX9, and ZFY genes, the individual has a female phenotype. This strongly suggests that the phenotypic sex in this case might be due to the involvement of other sex determining genes. We thank K M Gun for referring the case. Financial support of the Department of Biotechnology, Government of India, for creating Automated DNA Sequencing facility and providing support for carrying out the work is gratefully acknowledged. We are grateful to A J Rachel for her help in doing fluorescence in-situ hybridisation. 1
2
3 4 5
A 47,XXY female
Klinefelter HF, Reifenstein EC, Albright F. Syndrome characterized by gynecomastia, aspermatogenesis without a-Leydigism, and increased excretion of follicle stimulating hormone. J Clin Endocrinol 1942; 2: 615–27. Sinclair AH, Berta P, Palmer MS, et al. A gene from the human sexdetermining region encodes a protein with homology to a conserved DNA-binding motif. Nature 1990; 346: 240–44. Pevny LH, Lovell-Bedge. SOX genes find their feet. Curr Opin Genet Develop 1997; 7: 338–44. Cameron FJ, Sinclair AH. Mutation in SRY and SOX9: testisdetermining genes. Hum Mutation 1997; 9: 388–95. Sano K, Terashima K, Tanaka Y, Sasaki Y. Four cases of true hermaphroditism. Hinyokika Kiyo 1995; 41: 73–77.
Centre for Cellular and Molecular Biology, Hyderabad 500 007, India (L Singh); Institute of Reproductive Medicine, Calcutta; Centre for DNA Fingerprinting and Diagnostics, Hyderabad
Kumarasamy Thangaraj, Nalini J Gupta, Baidyanath Chakravarty, Lalji Singh
Klinefelter syndrome (47,XXY) with male phenotype is the most common sex chromosomal abnormality.2 It is believed that SRY (sex determining region on Y) is the major gene necessary to induce the undifferentiated bipotential gonadal primordium to develop as testis.2 Recently, several SRY-boxrelated (SOX) genes have been identified on autosomes.3 Mutations in the SRY or SOX gene have been implicated in sex reversal.4 We report an individual having 47,XXY chromosome constitution, normal SRY, SOX9, and ZFY genes and yet with a female phenotype. A 15-year-old phenotypic female has well developed breasts; prominent labioscrotal folds; an enlarged clitoris; feminine pubic hair distribution; urethra in the centre of the vestibule; a urogenital sinus 5 cm deep; a uterus; prepubertal in shape and size, palpable per rectum; and a cervix which could not be palpated. Ultrasonography of the abdomen revealed normal shape of urinary bladder with smooth and regular walls. Uterus was small in size, with normal shape and outline. Myometrium was homologous, cavity was empty, midline echo was normal. Laparoscopic assessment revealed a normal uterus. Right ovary was normal in size and shape with a cyst and a normal Fallopian tube. Left ovary was not seen as it was hidden beneath an epithelial fold. Chromosome analysis showed 47,XXY karyotype in all 100 metaphases analysed. Hormone profiles showed average female range of corticotrophin, T3, T4, thyrotrophin, and testosterone. Progesterone and prolactin were higher when compared with normal female levels. Histology of the right gonad showed features compatible with ovarian stroma. No
THE LANCET • Vol 352 • October 3, 1998
Acute neuropathy after exposure to sun in a patient treated with St John’s Wort Geoffrey M Bove
Extracts or whole-herb preparations of St John’s Wort (Hypericum perforatum) are popular to treat depression. Photosensitivity is known to be a possible side effect. We report one patient who, while taking St John’s Wort, developed subacute polyneuropathy after exposure to sun. A 35-year-old woman took ground whole St John’s Wort (500 mg/day) for mild depression, after reading a magazine article. After 4 weeks, she developed stinging pain on her face and dorsum of both hands (areas exposed to the sun). Spontaneous pain was mild but worsened during and after being in the sun. Pain was provoked by minimal mechanical stimuli such as light touch or air movement. Cooling increased and warming decreased the pain. She sought help when the same symptoms developed on her arms and legs a few hours after sunbathing, and were limited to the exposed skin. She was examined 2 weeks after the pain started. There were no skin burns on examination. Light brushing, a gust of air at room temperature, and cold (~5–10OC) were acutely painful and outlasted the stimulus for seconds afterwards, consistent with allodynia. These symptoms were restricted to
1121