Urinary 17α-hydroxyprogesterone in management of 21 -hydroxylase deficiency

Objectives: The study was designed to assess the reliability of measurement of 24-hour urinary 17α-hydroxyprogesterone (17-OHP) by radio-immunoassay (RIA) as an alternative biochemical assessment for monitoring the treatment of congenital adrenal hyperplasia (CAH) due to 21 -hydroxylase deficiency (21 -OHD) and to assess the need for sample purification by column chromatography to improve assay specificity.
Methodology: Morning serum 17-OHP was measured using RIA and 24-hour urinary pregnanetriol using gas chromatography. Twenty-four-hour urinary 17-OHP was measured in samples from 17 prepubertal patients with CAH due to 21 -OHD, and 20 normal prepubertal children as controls. In 24 urine samples, RIA of 17-OHP was performed with and without column chromatography.
Results: There was a good correlation between 24-hour urinary 17-OHP and 24-hour urinary pregnanetriol (r = 0.962, P <0.01) and between 24-hour urinary 17-OHP and morning serum 17-OHP ( r = 0.955, P <0.01). There was no significant difference in the RIA of the urine samples with and without purification by column chromatography.
Conclusions: The measurement of 24-hour urinary 17-OHP is a reliable alternative for the biochemical monitoring of 21-OHD, and RIA specificity is unaffected by omission of column chromatography.     

A Case of a Preterm Infant with 21-Hydroxylase Deficiency: Implications of the Biochemical Diagnosis with Urinary Pregnanetriolone by Gas Chromatography/Mass Spectrometry in Selected Ion Monitoring (GCMS-SIM)

The biochemical diagnosis of 21-hydroxylase deficiency (21-OHD) is difficult in preterm infants. To date, no marker for the biochemical diagnosis of 21-OHD has been found. Seventeen α-hydroxyprogesterone (17-OHP), is not useful because of interference by delta 5 steroids from the fetal adrenal cortex. A 5-d-old female infant, born at 31 wk of gestation, was suspected of having 21-OHD based on physical findings (mild clitoromegaly, pigmentation of the tongue and gingiva) as well as laboratory data (17-OHP >93.5 ng/ml by ELISA 7 prime extractive method in filter paper-dried blood spot and 718.3 ng/ml by RIA after high performance liquid chromatography extraction in serum; plasma ACTH 690 pg/ml; and serum testosterone 3,169 ng/dl). We examined her urinary steroid profiles by gas chromatography/mass spectrometry in selected ion monitoring (GCMS-SIM) at 8 d of age. The pregnanetriolone (Ptl) level was noticeably high (0.80 mg/g creatinine), which was strongly suggestive of 21-OHD. Gene analysis of CYP21A2 showed compound heterozygosity, one allele having a cluster mutation in exon 6 and the other having a large deletion including CYP21A2, confirming the diagnosis of 21-OHD. This case suggested that, in preterm infants, urinary Ptl by GCMS-SIM can be useful for the biochemical diagnosis of 21-OHD.     

21-羟化酶缺乏症青春期患儿治疗期指标监测

目的 监测21羟化酶缺乏(21-OHD)青春期患儿激素水平,筛选特异性较高的指标.方法 回顾分析21-OHD青春期患儿的临床资料.根据雄性化程度及骨龄将患儿分为控制较佳组和控制不佳组,对比两组间体质指数(BMI)、氢化可的松药物剂量及类固醇激素水平的差异;使用受试者工作特性(ROC)曲线定义控制不佳的截断值.结果 共纳...     

先天性肾上腺皮质增生症21羟化酶缺乏患者的生长与最终身高影响因素

先天性肾上腺皮质增生症(CAH)是一组常染色体隐性遗传病,由于肾上腺皮质激素合成酶的缺陷,皮质醇的合成部分或完全受阻使促肾上腺皮质激素(ACTH)分泌过多导致肾上腺皮质增生,同时皮质醇的前体产物过多堆积并转化为性激素.21羟化酶缺乏(21-OHD)是最常见的CAH,同时也是人类最常见的常染色体隐性遗传病之一,分为经典型...     

The ratio of second- and fourth-digit lengths and congenital adrenal hyperplasia due to 21-hydroxylase deficiency

Congenital adrenal hyperplasia (CAH) due to 21-hydroxylase deficiency results in excessive androgen exposure in the gestational period and various degrees of masculinization of the external genitalia in female foetuses. Intrauterine gonadal steroids are not only essential for the development of the genital organs but also affect some other extragenital organ development.

The second to fourth digit (2D/4D) ratio shows a sexually dimorphic pattern with longer fourth digit from second digit in men compared to women. A low 2D/4D ratio is associated with high sperm count, testosterone levels and reproductive success in men. A high 2D/4D ratio is associated with high oestrogen levels in women. Second and fourth digit ratio has also found to be correlated with sexual orientation, left hand preference autism and some adult onset diseases such as breast cancer and myocardial infarction.

We found lower 2D/4D ratio in female patients with 21-hydroxylase deficiency compared to healthy girls (p=0.000) and equal 2D/4D ratio for female patients when compared to male controls. Male patients with 21-hydroxylase deficiency had significantly lower 2D/4D ratio than female and male controls in the right hand. Healthy boys had lower 2D/4D ratio than healthy girls.

It is concluded that 2D/4D ratio established by intrauterine androgen levels influences the sexually dimorphic digit pattern.     

Non-classical 21-hydroxylase deficiency in children: association of adrenocorticotropic hormone-stimulated 17-hydroxyprogesterone with the risk of compound heterozygosity with severe mutations

AIM: To investigate the association between levels of 17-hydroxyprogesterone (17-OHP) and the risk of being compound heterozygous for severe mutations in children with non-classical 21-hydroxylase deficiency (NC21OHD). METHODS: In 86 Spanish NC21OHD children (75 families) an analysis of the 21-hydroxylase (21-OH) gene was performed by CYP21B-specific polymerase chain reaction amplification, allele-specific oligonucleotide hybridization and Southern blotting. Familial analysis established how the alleles segregated, and allowed the selection of 21-OH-genotyped normal and carrier children, which proved useful in determining a more precise definition of the cut-off for diagnosis. Receiver operating characteristics (ROC) curve analyses were performed to determine the potential value of 17-OHP in predicting compound heterozygosity for severe mutations. RESULTS: Thirty-four of the 86 children (39%) were found to carry one severe 21-OH mutation (7.3% deletions or conversions, 2.7% 655G, 2.7% Q318X, 1.3% 1172N, 1.3% R356W, and 3.3% double microconversions or small conversions involving single exons). The predominant mutation was V281L (56.7%). P453S and P30L were less frequent (3.3 and 2%). No patient showed two severe mutations. The degree of enzymic deficiency, as measured by basal or adrenocorticotropic hormone (ACTH)-stimulated 17-OHP levels in fully genotyped patients, but not clinical severity (age and number of symptoms at diagnosis), was found to be significantly greater in children with the severe/mild genotype. ROC curve analyses revealed a strong association between ACTH-17-OHP and genotype (area under the curve 0.908, SE 0.057). CONCLUSION: ACTH-stimulated 17-OHP may predict the risk of severe mutations in compound heterozygosity in children (maximum predictive value 93% sensitivity and 83% specificity for a cut-off at 151 nmol l(-1)), although a certain overlap in individual values is observed and performance of molecular analysis should never be obviated in the genetic counselling of these patients.     

Prenatal diagnosis and treatment of steroid 21-hydroxylase deficiency (congenital adrenal hyperplasia)

Steroid 21-hydroxylase deficiency congenital adrenal hyperplasia is the most common cause of genital ambiguity in females at birth. Inhibited formation of cortisol causes increase in the release of ACTH in turn leading to overproduction of adrenal androgens. This predisposes the affected female fetus to prenatal development of genital ambiguity. A large number of patients also have aldosterone insufficiency which manifests after birth in form of inability to conserve sodium and to excrete potassium which can lead to adrenal shock and neonatal death, if left untreated. Prenatal diagnosis is possible using several methods like steroid assay of amniotic fluid and,HLA typing. Recently with advancement more accurate direct molecular genetic techniques have been utilized on chorionic villus samples in first trimester of pregnancy. Prenatal treatment is also possible and pregnancies can be managed by administering dexamethasone to the mother as soon as pregnancy is diagnosed. This suppresses fetal androgen production in genetic females preventing virilization and leading to normal development. Prenatal diagnosis and treatment are highly desirable in families with a positive family history towards birth of a child without features of the disease.     

Urinary excretion of 17-hydroxypregnanolones in patients with different forms of congenital adrenal hyperplasia due to steroid 21-hydroxylase deficiency

To improve diagnostic criteria in different (classical salt-wasting (SW), classical simple virilizing (SV) and non classical late onset (LO)) forms of congential adrenal hyperplasia (CAH) due to steroid 21-hydroxylase deficiency, we investigated the urinary excretion of 17-hydroxypregnanolones (17OH-PO(5) and (5), 15-hydroxypregnanolone(15OH-PO), pregnanetriol(PT) and 11-oxo-pregnanetriol (11-O-PT) compared to hydrocortisone metabolities During the 1st month of life newborn infants with CAH-SW excreted from barely detectable to very large amounts of 17OH-PO(5), 15OH-PO and PT, and, in 12 of 14 cases, also 11-O-PT in their urines. From the 1st to the 28th day of life, cortisol metabolites were virtually absent in urines of CAH-SW infants. This was in contrast of 36 healthy newborn infants. We measured the excretion of 17OH-PO(5) in children with CAH of whom 19 patients with CAH-SV had a median 17OH-PO(5) excretion of 1110 g/day (range: 152–5515). In 21patients with CAH-LO, median excretion of 17OH-PO(5) was 294g/day (range: 66–1273). Besides the conventional metabolites of 17-hydroxyprogesterone (17OH-PO(5), PT and 11-O-PT),no 17OH-PO(5) was detected in the urines of 14 patients with precocious pubarche, in 14 patients with virilization of unknown origin and in 94 healthy children of comparable age. The ratio of 17OH-PO(5) to tetrahydrocortisone (THE) discriminated between CAH-SV and CAH-LO from the 1st to the 18th year of age. The determination of urinary 17OH-PO(5) is an excellent diagnostic method in CAH-SV as well as CAH-LO.     

Temporal and individual variations in the dose of glucocorticoid used for the treatment of salt-losing congenital virilizing adrenal hyperplasia due to 21-hydroxylase deficiency

The dose of glucocorticoid was evaluated in the treatment of 19 patients with salt-losing congenital adrenal hyperplasia due to complete or nearly complete 21-hydroxylase deficiency. In most cases, follow-up was from infancy to puberty. The dose of steroid was expressed as oral cortisol (mg/m² body surface area 124 hours); the equivalent doses of the various glucocorticoid preparations was as follows: 100 mg oral cortisol = 120 mg oral cortisone acetate = 25 mg oral prednisone = 50 mg intramuscular cortisol = 60 mg intramuscular cortisone acetate. The dose of glucocorticoid producing good laboratory and clinical control varied significantly with age. The dose fell from 26 mg/m²/24 hours in early infancy to 19 mg/m²/24 hours between 6 and 8 years of age, and then rose to 23–24 mglm²/hour in adolescence. In addition to these age-related changes, there were large individual variations at each age. Indeed, the values from 4 of the 19 patients were not included in the calculation of the mean because they were more than 3 SD either above or below the mean. For the rest of the patients, the coefficient of variation ranged from 14.5% to 37.2%. It is concluded that glucocorticoid therapy must be adjusted carefully to the age and needs of each patient.     

Detection of late onset steroid 21-hydroxylase deficiency by capillary gas chromatographic profiling of urinary steroids in children and adolescents

Patients suffering from late onset 21-hydroxylase deficiency (LO-CAH) excreted only slightly higher amounts of 17-hydroxypregnanolone (17-OH-PO), pregnanetriol (PT) and 11-oxo-pregnanetriol (11-O-PT) than age-matched healthy controls. To discriminate between LO-CAH and virilization of unknown origin and precocious pubarche, we calculated the following ratios: (1) pregnanetriol to tetrahydrocortisone (PT/THE), (2) the sum of 17-OH-PO, PT and 11-O-PT (OHP-M) to the sum of THE, tetrahydrocortisol (THF) and allotetrahydrocortisol (a-THF) (C-M) and (3) 11-O-PT to C-M. The following patients were studied: 9 patients with congenital adrenal hyperplasia due to 21-hydroxylase deficiency-non-salt losing (CAH-NSL), never treated; 8 patients with CAH (NSL/SL: 3/5) off treatment; 10 patients with LO-CAH; 11 patients with virilization of unknown origin (prepubertal/pubertal: 5/6) and 9 patients with precocious pubarche. Healthy individuals and obligatory heterozygote carriers of comparable ages served as controls. LO-CAH showed increased ratios (median (range)) of PT/THE: 2.27, (1.15–9.09), OHP-M/C-M: 2.30, (1.24–8.15), and 11-O-PT/C-M: 0.24, (0.13–1.23) compared to healthy individuals and heterozygous carriers: PT/THE 0.28, (0.03–0.57), OHP-M/C-M 0.23, (0.06–0.46) and 11-O-PT/C-M<0.01, (<0.01–0.06), respectively. The calculation of ratios, rather than absolute amounts seems to allow the detection of LO-CAH in a single spontaneously voided urine specimen. The clinical and measurable hormonal manifestations of LO-CAH occur at the same time.Abbreviations LO-CAH late onset 21-hydroxylase deficiency - CAH congenital adrenal hyperplasia due to 21-hydroxylase deficiency - SL salt losing - NSL non-salt losing - p.p. prepubertal - p. pubertal - 17-OH-PO 17-hydroxypregnanolone (5-pregnane-3,17-diol-20-one) - PT pregnanetriol (5-pregnane-3,17,20-triol) - 11-O-PT 11-oxo-pregnanetriol (5-pregnane-3,17,20-triol-11-one) - THE tetrahydrocortisone (5-pregnane-3,17,21-triol-11,20-dione) - THF tetrahydrocortisol (5-pregnane-3,11,17,21-tetrol-20-one) - a-THF allotetrahydrocortisol (5-pregnane-3,11,17,21-tetrol-20-one) - OHP-M 17-OH-PO+PT+11-O-PT - C-M THE+THF+a-THF Dedicated to Prof. Enno Kleihauer, Ulm, on the occasion of his 60th birthday     

Adiponectin levels are high in children with classic congenital adrenal hyperplasia (CAH) due to 21-hydroxylase deficiency

Objective: It has been shown that adiponectin serves as an insulin-sensitizing adipokine. Serum concentrations of adiponectin are low in children with obesity, and increase with fat mass loss, indicating that adiponectin can serve as a biomarker. Since the prevalence of overweight and obesity is increased in children with congenital adrenal hyperplasia (CAH), our study aimed to evaluate serum levels of adiponectin in a cohort of CAH children and adolescents, and their associations with clinical parameters such as chronological age (CA), body mass index (BMI), Tanner stage (TS), medication and metabolic control.
Patients and methods: We studied 51 patients, aged between 5.6 and 19.6 years (median 11.8; 30 females, 21 males), cross-sectionally. All patients had genetically confirmed CAH and received standard steroid substitution therapy. Adiponectin was measured by an enzyme linked immunoassay. Since BMI SDS of the CAH cohort were significantly higher compared to the reference population, we built matched pairs with healthy Caucasian subjects from a normal representative cohort for sex, Tanner stage, chronologic age and BMI.
Results: Adiponectin concentrations were significantly higher in CAH patients (median 11 μg/L) compared to the matched controls (6.7 μg/L, p < 0.0001). Correlation analyses in CAH patients revealed a significant inverse relationship between adiponectin and CA, TS, BMI, serum DHEAS and serum testosterone, but no correlation with hydrocortisone and fludrocortisone dosage.
Conclusion: Currently, the importance of the elevated adiponectin concentrations in CAH children for risk assessment is not clear. However, our data imply that besides adequate metabolic control of glucocorticoid substitution, a long-term follow-up of other metabolic markers of insulin resistance should be conducted in CAH patients.     

Salt-wasting 21-hydroxylase deficiency congenital adrenal hyperplasia and pyloric stenosis in two Hispanic brothers

The differential diagnosis of vomiting and dehydration in the first month of life includes congenital adrenal hyperplasia (CAH) and pyloric stenosis (PS). Each diagnosis may mask the presence of the other, requiring careful evaluation and follow-up. We document the occurrence of CAH and PS in two Hispanic siblings.     

Abnormal steroidogenesis in three patients with Antley–Bixler syndrome: Apparent decreased activity of 17α-hydroxylase, 17,20-lyase and 21-hydroxylase

BACKGROUND: Antley-Bixler syndrome (ABS) is characterized mainly by abnormal skeletal morphogenesis such as craniosynostosis and radiohumeral synostosis, and by ambiguous genitalia in some cases. The mechanisms resulting in these deformities have not been determined. Methods: The adrenal and gonadal function of three Japanese ABS patients were evaluated. Patient 1 (17-year-old-male) had bilateral cryptoorchidism, delayed puberty and symptoms of glucocorticoid deficiency. Patient 2 (14-year-old male) and patient 3 (4-year-old female) presented with emaciation. Additionally, patient 3 had partial labial fusion and common urogenital sinus. In each patient, blood sampling for steroid analysis before and after rapid adrenocorticotropic hormone (ACTH) stimulation was carried out. Additionally, urinary steroids were quantified. Molecular analysis of CYP17 and CYP21A2 were also performed. Results: All patients showed elevated basal 17alpha-deoxysteroid levels. Although the 17alpha-deoxysteroid levels further increased after rapid ACTH stimulation, 17alpha-hydroxysteroids including cortisol did not respond, suggesting impaired 17alpha-hydroxylation. Patient 1 and patient 2 showed low adrenal androgen blood levels both before and after rapid ACTH stimulation. Patient 3 showed lower than normal excretions of urinary androgens. Additionally, a prolonged ACTH stimulation in patient 3 failed to elicit significant increase of adrenal androgens. These findings suggested impaired 17,20-lyase activity. In contrast to attenuated 17alpha-hydroxycorticosteroids, notably cortisol, elevated 17alpha-hydroxyprogesterone (17OHP) levels were observed, not only in pubertal patients (1 and 2) but also in prepubertal patient 3, indicating impaired 21-hydroxylation. This assumption was supported by increased urinary 21-deoxycortisol metabolite excretion in patients 2 and 3. With the exception of a heterozygous mutation of CYP17 in one of the patients, other mutations of this gene or CYP21A2 were identified in any of the patients. CONCLUSION: Combined decreased 17alpha-hydroxylation, 17,20-lyase activity and 21-hydroxylation was detected in three ABS patients. Considering that the enzymes responsible are all cytochrome P450 enzymes and that another cytochrome P450 enzyme, lanosterol 14alpha-demethylase, has recently been shown to be impaired in an ABS patient, we speculate that dysfunction of a system which commonly regulates cytochrome P 450 activity may be responsible for the ABS phenotype.     

Mutation distribution and CYP21/C4 locus variability in Brazilian families with the classical form of the 21-hydroxylase deficiency

Deficiency of adrenal steroid 21-hydroxylase is the most common form of congenital adrenal hyperplasia and it is considered to be responsible for 90% of the disease. This paper describes for the first time the CYP21B mutation profile in Brazilian patients. We genotyped 41 families with at least one individual affected with the classical form of the 21-hydroxylase deficiency, representing 74 unrelated alleles. In order to characterize different disease-causing alleles, genotyping was performed by Southern blot analysis with three restriction enzymes, allele-specific oligonucleotide hybridization, and allele-specific PCR. Different alleles were distinguished by TaqI C4B RFLP, gene duplications or deletions of either CYP21A + C4B or CYP21B + C4B, large gene conversions and eight mutations that might have been introduced into CYP21B from CYP21A by microconversion events. At least one mutation was detected in 24 different disease-causing alleles, which represents about 85% of the affected alleles in those families. The frequency of the 30 kb deletion of CYP21B was lower than that described for Caucasians. The mutation Sp2 showed the highest frequency (24.65%) and was present mainly in salt-wasting patients, although it was also detected in some patients with the simple virilizing form of the disease. Conversely, I172N showed a frequency of 18.91% and was found mostly in patients affected with the simple virilizing form of the disease. Five other mutations were determined at low frequency, but CL6 was not found in any of the tested alleles.     

Molecular detection of genetic defects in congenital adrenal hyperplasia due to 21-hydroxylase deficiency: A study of 27 families

Congenital adrenal hyperplasia (CAH) due to 21-hydroxylase (21-OHase) deficiency is inherited as an autosomal recessive trait. Patients can present with the salt wasting, simple virilizing or a non-classical form of the disease. The gene for P450C21, the enzyme carrying 21-OHase activity, has been mapped to the major histocompatibility complex on chromosome 6p. Using molecular hybridisation techniques we have studied the genetic defect in 27 families with one or more affected off-spring diagnosed and treated at the University Hospital of Essen. DNA samples were digested with restriction endonucleaseTaqI,PvuII,BglII, andEcoRI and analysed by Southern blot hybridisation with the cDNA probe pC21/3c. Eleven of 40 haplotypes associated with the salt wasting form were found to have a large deletion of 30 kb affecting the 5 end of the active 21-OHase gene and the 3 end of the closely linked pseudogene. Results in another 11 cases are compatible with gene conversion; 18 cases were not informative. The 30 kb deletion was associated with a combination of the HLA antigens Bw47 and DR7 in 7 of 11 cases. In the haplotypes with gene conversion, no linkage disequilibrium to HLA antigens was found. No apparent gene alterations were detected in simple virilizing and non-classical haplotypes. The direct detection of the genetic defect in 55% of the salt wasting haplotypes may help to improve predictive testing in families with CAH.     

Congenital adrenal hyperplasia due to 11 β-hydroxylase deficiency in Saudi Arabia: clinical and biochemical characteristics

Over a 10-year-period, 78 Saudi children with congenital adrenal hyperplasia were seen at King Khalid University Hospital, Riyadh. Of these, 20 (25.6%) patients from 11 families were 11/3-hydroxylase deficient. Their mean age was 2.8 years (range 0-10 years). The clinical expression was somewhat severe; pseudoprecocious puberty in males and variable degrees of virilization in females which led to wrong sex assignment in seven (58.3%). Three patients had neonatal salt-wasting before treatment. Moderate to severe hypertension associated with hypokalaemia was present in another six. In four siblings hypertension persisted inspite of adequate hydrocortisone therapy. It is concluded that congenital adrenal hyperplasia due to 11β-hydroxylase deficiency is relatively frequent among the Saudi Arabian population. In view of the severity of the clinical expression and complications, physicians should be aware of the disease and have a high index of suspicion in order to detect and treat such patients early enough to avoid or minimize the unwanted sequelae.     

Value of direct measurement of active renin concentrations in congenital adrenal hyperplasia due to 21-hydroxylase deficiency

In congenital adrenal hyperplasia (CAR) due to 21-hydroxylase deficiency, measurement of plasma renin activity (PRA) has been the method of choice in diagnosing salt loss and in monitoring adequacy of mineralocorticoid replacement therapy. Due to methodological problems in PRA determinations, direct immunoradiometric assays for the measurement of active renin concentration have been developed. We measured PRA and active renin concentrations simultaneously in 39 patients with CAH (30 salt-wasting, 9 simple virilizing) to evaluate the potential role of this new method in the management of this disease. PRA was determined with an enzymatic assay (sample volume: 2 × 1000 l plasma), active renin concentration with a direct immunoradiometric assay (sample volume: 2 × 200 l plasma or serum). We found a highly significant correlation between active renin and PRA in our patients (P < 0.001), as previously shown in healthy subjects. Active renin was as reliable as PRA to assess the quality of mineralocorticoid replacement.     

Family studies of the steroid 21-hydroxylase and coplement C4 genes define 11 haplotypes in classical congenital adrenal hyperplasia in The Netherlands

Two steroid 21-hydroxylase genes are normally present within the human major histocompatibility complex near the genes encoding the fourth component of complement (C4A and C4B). Steroid 21-hydroxylase is encoded by the CYP21 gene, while the highly homologous CYP21P gene is a pseudogene. We studied steroid 21-hydroxylase and complement C4 haplotypes in 33 Dutch patients (29 families) suffering form classical congenital adrenal hyperplasia (CAH) and in their 80 family members, and also in 55 unrelated healthy controls, using 21-hydroxylase and complement C4 cDNA probes. Eleven different haplotypes, defined in terms of gene deletions, gene duplications, conversions of CYP21 to CYP21P, and long and short C4 genes, were found. In 23% of the patients' haplotypes, the CYP21 gene was deleted; in 12%, it was converted into a CYP21P pseudogene. In the remaining 65%, the defect was apparently caused by a mutation not detectable by this method. The most common haplotype (with one CYP21 and one CYP21P gene) was significantly more often observed in patients with simple virilizing CAH than in those with salt-losing CAH. Comparison of the 21-hydroxylase haplotypes found in CAH patients from several countries shows evidence for considerable genetic variation between the groups studied.