Diagnóstico y tratamiento prenatal de la hyperplasia suprarenal congénita por déficit clásico de 21 hidroxilasa / Prenatal diagnosis and treatment of congenital adrenal hyperplasia due to classical deficiency of 21 hydroxulase

La forma más frecuente de hyperplasia suprarrenal congénita se debe al déficit de enzima 21 hidroxilasa que en su forma grave produce ambigüedad genital en la niña afecta. Este déficit puede ser diagnosticado intraútero mediante el análisis genético del ADN fetal obtenido de las vellosidades coriónicas hacia la 9-10 semana de gestación. La virilización puede ser eficazmente prevenida mediante la administración de dexametasona a la madre antes de la 7-8 semana de gestación. Solo se debe continuar el tratamiento en la niña afecta de forma grave. Los datos existente muestran que el diagnóstico y tratamiento prenatal son seguros tanto para la madre como para el feto. Todos los sujetos tratados deben ser seguidos hasta la vida adulta para valoración de su desarrollo físico, cognitivo y emocional (AU)

The most common form of congenital adrenal hyperplasia is 21-hydroxylase deficiency which in its sever form can cause genital ambiguity in females. Steroid 21-OHD can be diagnosed in utero through molecular genetic analysis of fetal DNA form a chorionic villous biopsy in the 10th week of gestation. Appropriate prenatal treatment by dexametasone administration to the pregnant mother is effective in reducing genital virilizaton in the female fetus. Data from current studies show that prenatal diagnosis and treatment are safe for the mother and the fetus. All fetus exposed to dexametasone should have their physical, cognitive and emotional developments recorded (AU)

Monogenic and polygenic models detected in steroid 21-hydroxylase deficiency-related paediatric hyperandrogenism.

AIMS: Hyperandrogenism, although mostly due to polygenic interactions, is monogenic for some enzymatic adrenal deficiencies. This study evaluates mono- and biallelic 21-hydroxylase deficiency (21OHD)-related hyperandrogenism in pediatric patients. Sensitizing and protective polymorphisms were investigated in carriers and cryptic forms of 21OHD. METHODS: The study involved a monogenic analysis of CYP21A2 in patients (375 nonclassical 21OHD [NC21OHD] children; 306 hyperandrogenic 21OHD carriers, n = 306) and a polygenic association study (CAPN10-UCSNP44, PON1-108, TNFR2-M196R, IGF2-ApaI and IRS1-G972R polymorphisms) of 170 hyperandrogenic carriers plus 277 family members (control groups). The metabolic marker 17OH progesterone defined the degree of deficiency; clinical expressivity was determined by pediatric endocrinologists. RESULTS: The group of 21OHD carriers manifesting hyperandrogenism was enriched in the CAPN-UCSNP44 rare variant in homozygosity (4.9 vs. 0.4%, NCBI data for the general population; p = 0.004). In our patients and controls, contrasting distributions were observed for this and another polymorphism, TNFR2-196R. In a recessive model, their rare variants were more frequently detected among the forms with high (p = 0.048) and low (p = 0.034) expressivity respectively. CONCLUSIONS: 21OHD-related pediatric hyperandrogenism follows monogenic and polygenic models. The opposite behaviors in terms of clinical expressivity detected for CAPN-UCSNP44 and TNFR2-M196R rare variants suggest these variants to be sensitizing and protective factors respectively in adrenal hyperandrogenism.

Gene duplications in 21-hydroxylase deficiency: the importance of accurate molecular diagnosis in carrier detection and prenatal diagnosis.

BACKGROUND: The detection of 21-OH deficiency (21OHD) carriers in the general population requires that misinterpretations of apparently severe mutations in alleles carrying duplicated genes be avoided. Prenatal treatment prevents virilization in female fetuses and genetic counseling may be offered to couples in which one partner is either a patient or a carrier. This paper proposes a semiquantitative PCR method involving primer extension that distinguishes the severe point mutation Q318X in single gene copy alleles from the normal/nondeficient variant in gene-duplicated alleles. SAMPLES AND METHODS: DNA from 65 individuals carrying Q318X variants, that of 85 partners of 21OHD carriers or patients, and one fetal sample (as well as the DNA of his family) were analyzed. 21OHD alleles were studied by gene-specific PCR/allele-specific oligonucleotides hybridization for common mutations, Southern analysis, complementary direct sequencing and microsatellite typing. Primer extension analysis of the Q318X variants using fluorescent dideoxynucleotides was performed on CYP21A2 gene-specific PCR-amplified DNA samples from controls, patients, potential carriers and prenatal samples. RESULTS: Different fluorescence patterns were seen for the severe mutation (single gene copy) and the nondeficient (gene-duplicated) alleles carrying Q318X. The normal/mutant fluorescence peak (N/M) ratio was < 1 in all heterozygous carriers (mean 0.83; min. 0.70; max. 0.95). In all normal individuals carrying the gene-duplicated Q318X normal variant, the N/M ratio was > 1 (mean 1.69; min. 1.44; max. 2.02). CONCLUSION: The proposed method discriminated between the severe Q318X mutation and the normal Q318X variant in gene duplication, and could be a useful complementary tool in prenatal diagnosis and carrier detection.

Déficit de 21-hidroxilasa: aspectos actuales / 21-hydroxylase deficiency: current aspects

La hiperplasia suprarrenal congénita (HSC) es una de las alteraciones autosómicas recesivas más frecuentes. Caracterizada por un defecto enzimático en la síntesis de cortisol, la causa es, en el 95% de los casos, la deficiencia de la enzima 21-hidroxilasa (21-OH). La 17-OH progesterona, precursor del cortisol, presenta valores elevados, marcadores del diagnóstico. Esta enfermedad presenta diferentes formas clínicas; las clásicas o graves comienzan desde el período neonatal, con síntomas debidos al exceso de andrógenos suprarrenales como virilización y ambigüedad de los genitales externos de las niñas afectadas. En más del 70% de los casos se asocia pérdida salina (deficiencia de aldosterona), potencialmente letal en varones que no se diagnostican precozmente. Resumimos las diferentes formas de presentación de la deficiencia de 21-OH, y describimos el diagnóstico y el tratamiento con gluco y mineralcorticoides, con especial énfasis en la importancia de utilizar dosis de estrés de hidrocortisona, cuando es necesario. Los avances quirúrgicos actuales ofrecen una corrección funcional de las pacientes afectadas. Los programas de detección precoz evitan la asignación incorrecta de sexo en la recién nacida y pueden salvar la vida de los varones con formas graves y pérdida salina. Comentamos el diagnóstico genético-molecular del CYP21A2 (cromosoma 6p 21.3) y las características en la población española. Revisamos las directrices futuras para el estudio y el tratamiento de esta enfermedad, incluyendo diversos tratamientos como la flutamida, la hormona de crecimiento, los antagonistas de las gonadotropinas o la relación con el síndrome de ovario poliquístico. El diagnóstico y el tratamiento prenatales del feto femenino afectado son posibles, y los resultados son alentadores. Comentamos, también, el abordaje hacia la transición y la edad adulta, y la relevancia del control de la mujer con HAC durante la gestación

Congenital adrenal hyperplasia (CAH) is one of the most frequent autosomal recessive disorders. It is characterized by a deficiency of an enzyme involved in cortisol synthesis and in 95% of patients the cause is 21-hydroxylase deficiency. A diagnostic marker is elevated levels of 17-hydroxyprogesterone, a precursor of cortisol. CAH has several clinical forms, and classical or severe forms manifest in the neonatal period with symptoms due to excess adrenal androgen production such as virilization and ambiguity of the external genitalia in affected girls. In more than 70% of patients, there is associated salt wasting (aldosterone deficiency), which can be fatal in males without an early diagnosis. We summarize the various forms of presentation of 21-hydroxylase deficiency and describe diagnosis and treatment with gluco- and mineralocorticoids, with special emphasis on the importance of using stress doses of hydrocortisone when necessary. Current surgical advances provide functional correction in affected patients. Screening programs avoid incorrect sex assignment in the newborn and can save the lives of males with severe forms and salt wasting. We discuss the genetic-molecular diagnosis of CYP21A2 (chromosome 6p 21.3) and its characteristics in the Spanish population. We review future recommendations for the study and management of this disease, including several treatments such as flutamide, growth hormone, and gonadotrophin antagonists, as well as the association with polycystic ovary syndrome. Prenatal diagnosis and treatment in affected female fetuses are feasible and the results are encouraging. We also discuss the management of the transition to adulthood and the importance of follow-up of women with CAH during pregnancy

Neonatal salt-wasting and 11 beta-hydroxylase deficiency in a child carrying a homozygous deletion hybrid CYP11B2 (aldosterone synthase)-CYP11B1 (11 beta-hydroxylase).

This article reports the case of a boy diagnosed at 1.8 years of age with congenital adrenal hyperplasia due to 11 beta-hydroxylase deficiency. The patient showed salt-wasting episodes during the neonatal period. On molecular analysis, a homozygous deletion hybrid (CYP11B2-CYP11B1) involving the CYP11B locus at 8q24.3 was found. Southern blot analysis showed the break point of the chimera gene to be located before intron 5; sequence analysis identified it at exon 4 between codons 202 and 248. This CYP11B2(5')/B1(3') hybrid should lack aldosterone synthase activity (due to the CYP11B1 residues at exons 5 and 6), and the enzyme it codes for should not be promoted by adrenocorticotropic hormone (ACTH) (CYP11B2 promoter sequences). The patient phenotype - neonatal salt-wasting and 11 beta-hydroxylase deficiency - is in agreement with this hybrid structure. This is the first time a homozygous deletion hybrid generated by unequal crossover has been described in exon 4. This genetic lesion appears to be the reciprocal product from the recombination event that causes glucocorticoid-remediable aldosteronism, a duplication dominant allele (CYP11B2-CYP11B1/B2-CYP11B1) coding for additional aldosterone synthase activity regulated by ACTH. The clinical presentation of the condition in this patient contributes to the in vivo understanding of the regulation of this complex locus in which two 'duplicated' genes have evolved different regulatory and enzymatic activities involved in mineralocorticoid and glucocorticoid synthesis in the adrenal glands. The fact that this allele was first predicted and has now been documented clinically and molecularly in vivo is particularly noteworthy.

Clinical manifestations and hormonal profile of two women with Cushing's disease and mild deficiency of 21-hydroxylase.

The development of Cushing's disease among patients with deficiency of 21-hydroxylase has not been observed to date. The clinical manifestations and the hormonal profile of this exceptional association are herein described through the study of two cases. The first one was a 39-yr-old woman who had undergone non-curative transsphenoidal surgery for a pituitary-dependent Cushing's syndrome 12 yr before. She showed hypertension, central obesity, severe hirsutism, alopecia and hyperpigmentation. Urinary excretion of cortisol was normal, but ACTH levels were very high and hormonal dynamic studies (cortisol circadian rhythm, insulin-induced hypoglycemia and dexamethasone suppression tests) revealed the qualitative disturbances that characterize Cushing's disease. Serum concentrations of androstenedione, free testosterone and 17-hydroxyprogesterone were clearly increased. Reexamination of the tissue samples from previous surgery confirmed the presence of an ACTH-producing pituitary adenoma. CYP21 gene analysis found the splicing 655G mutation at intron 2 and the V281L mutation at exon 7. The second case was a 21-yr-old woman who was diagnosed with pituitary ACTH-dependent Cushing's syndrome according to unequivocal clinical and laboratory findings. However, hirsutism was particularly severe and both serum androgens and 17-hydroxyprogesterone were elevated. The patient was heterozygote for a large conversion of CYP21 gene. In these cases, the clinical and biochemical expression of Cushing's syndrome was determined by the different severity of 21-hydroxylase deficiency and the subsequent residual ability of adrenal cortex to synthesize cortisol.

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.

Gene conversion (655G splicing mutation) and the founder effect (Gln318Stop) contribute to the most frequent severe point mutations in congenital adrenal hyperplasia (21-hydroxylase deficiency) in the Spanish population.

This study addresses the contributions of gene conversion and a founder effect to the distribution of the two most frequent severe point mutations of the 21-hydroxylase (21OH) gene causing congenital adrenal hyperplasia: the 655G splicing mutation at intron 2, and Gln318Stop in a Spanish population. Direct and indirect analyses of segregated mutant and normal 21OH genes in 200 Spanish families (classic and nonclassic 21OH deficiency) were performed. Both mechanisms were found to contribute to different degrees to the defective investigated alleles. The 655G splicing mutation (62 alleles, 15.5%) seemed to be almost exclusively related to recent conversion events, whereas Gln318Stop (33 alleles, 8.3%) is more likely to be due to the dissemination of remotely generated mutant alleles. Other severe defective alleles, 8 bp-deletion (13 alleles, 3.3%), 306insT (5 alleles, 1.3%), and gene deletions (43 alleles, 11%), as well as the mild mutation Val281Leu (120 alleles, 30%), also appear to be strongly associated with particular D6S273 alleles. Although gene conversion contributes to the generation of severe 21OH alleles, the high frequency of some severe mutations in different geographic areas is consistent with a founder effect.

Deletion of the long arm of the Y chromosome in an adolescent with short stature and hypogonadism.

We describe a patient with short stature more than that expected for non-treated congenital adrenal hyperplasia due to nonclassic 21-hydroxylase deficiency with deletions in the long arm of the Y chromosome including the CGY gene and the AZF subregions.

A novel frameshift mutation in the first exon of the 21-OH gene found in homozygosity in an apparently nonconsanguineous family.

Congenital adrenal hyperplasia is most frequently due to steroid 21-hydroxylase (21-OH) deficiency. Due to the existence of a pseudogene in tandem duplicated with the 21-OH gene, asymmetric recombination causes the majority of the molecular defects underlying this deficiency: gene conversions and deletions of the functional gene. Screening for a small array of mutations, those existing in the pseudogene together with deletions, allows the characterization of most mutated alleles, 91% in the Spanish population. We report the case of a boy from a nonconsanguineous family, diagnosed during the neonatal period of a salt-wasting form of the deficiency, in which this screening did not allow the characterization of the paternal or the maternal allele. This infrequent finding in a nonconsanguineous family was further investigated. Single-strand conformation polymorphism screening for new mutations revealed an abnormally migrating pattern when polymerase chain reaction fragments from 21-OH gene exon 1 of the patient and relatives were analyzed. Upon direct sequencing, the insertion of a T at position 64 (64insT, frameshift generating a stop codon at exon 2) was found in homozygosity in the patient. Microsatellite typing in the HLA region revealed the patient to be homozygous for five markers (heterozygosities 0.62 to 0.74). Apparently this new mutation was generated several generations ago and has been preserved for years. Consanguinity had been discarded for several generations, although both families could be traced back to a small rural area in Navarra (Spain).

Microsatellite markers in the indirect analysis of the steroid 21-hydroxylase gene.

Prenatal diagnosis and treatment of congenital adrenal hyperplasia due to steroid 21-hydroxylase (21-OH) deficiency has been proved to be effective. Screening for a panel of nine point mutations, deletions, and gene conversions allows the identification of most of the mutations, although 6.12 per cent of chromosomes remain uncharacterized. In the present study, microsatellite typing in the HLA region was performed in 23 21-OH deficiency families to determine the usefulness of these markers in the indirect identification of disease alleles. Two Généthon markers (D6S273 and D6S439) in the HLA complex, class III and II regions in 5' and 3', respectively to the CYP21 gene, were typed together with a microsatellite at intron 3 of the TAP1 gene also in 3'. The heterozygosity of these markers provided informativity in all but on family, in which only the father was informative. Direct genotyping of the chromosomes confirmed in each case the correct assignment of the disease alleles in the sibling. The indirect analysis of the 21-OH gene through D6S273, TAP1, and D6S439 microsatellites provides useful information in the molecular analysis of steroid 21-OH deficiency.

[Characterization of steroid 21-hydroxylase gene mutations in a oligosymptomatic form of congenital adrenal hyperplasia: family study]. / Caracterización de las mutaciones del gen de la esteroide 21-hidroxilasa en una forma oligosintomática de hiperplasia suprarrenal congénita: estudio familiar.

BACKGROUND: 21-hydroxilase deficiency accounts for over 90% of all cases of congenital adrenal hyperplasia (CAH). There is a non-negligible incidence of both severe and nonclassical forms of this genetic disorder. Enzyme deficiency is due to mutations in the gene encoding adrenal 21-hydroxylase (named CYP 21B) and is inherited in an autosomical recesive way. Complete or partial impairment of enzyme activity has been correlated with the different clinical forms of the disease. PATIENTS AND METHODS: In the present paper CYP 21B gene analysis results obtained in a family with two kindred affected by a nonclassical form of the disease are shown. Clinical assessment of these two kindred showed a very mild form of the disease, whereas biochemical results suggested a late-onset partial 21-hydroxylase deficiency. Genotyping for deletions and 10 point mutations in the CYP 21B gene was performed by Southern blot analysis and polymerase chain reaction (PCR) allele-specific oligonucleotide (ASO) hybridation technique. RESULTS: Molecular genetic analysis performed in the two affected patients and two further relatives allowed us to detect the presence of different mutations in the two alleles of the CYP 21B gene. One of these mutations was severe (655G) and came from maternal line, whereas the other was mild (Val281Leu) and originated in paternal line. CONCLUSION: Molecular genetic analysis allows the possibility of finding severe (and non-expected) mutations in patients with clinically mild and late-onset forms of the 21-hydroxylase deficiency.

[Prenatal molecular genetic diagnosis and treatment of congenital adrenal hyperplasia due to 21-hydroxylase deficiency]. / Diagnóstico genético molecular y tratamiento prenatal de la hiperplasia adrenal congénita por déficit de la enzima 21-hidroxilasa.

Congenital adrenal hyperplasia due to 21-hydroxylase deficiency (21-OH), in its severe forms, produces virilization of the external genitalia of the affected female fetus. Early treatment with dexamethasone of the pregnant mother at risk of a fetus with 21-OH deficiency avoids the masculinization of the affected female fetus. We present a pregnant mother, where the prenatal diagnosis was established by DNA analysis of a chorionic villous sample obtained in the 9th week of gestation. Molecular analysis showed the female fetus to be affected of 21-OH deficiency. Maternal treatment with dexamethasone started on the 6th week of gestation has prevented the virilization of the affected baby. No significant side effects have been encountered. Prenatal diagnosis and treatment for 21-OH deficiency is effective and safe, as is described in the literature. This is the first case in Spain where both prenatal molecular diagnosis and treatment for 21-OH deficiency have been reported.

Analysis of steroid 21-hydroxylase gene mutations in the Spanish population.

Steroid 21-hydroxylase deficiency is the major cause of congenital adrenal hyperplasia. Genotyping for deletions and nine point mutations in the CYP21 gene has been performed in 38 Spanish patients and their relatives by Southern blot analysis and allele-specific oligonucleotide hybridization. Three clinical variants were included in this study, viz., salt-wasting (SW, 21 patients), simple virilizer (SV, two patients), and late-onset (LO, 15 patients) forms. Twenty-three patient genotypes (16 SW, two SV, and five LO) were fully characterized. In both alleles, all but one of these severe forms (SW and SV) presented mutations that abolished or severely affected enzymatic activity. Patients with LO forms showed mutations that moderately impaired enzymatic activity in both alleles, or severe mutations in only one chromosome. Of 46 chromosomes from severe forms, 41 were characterized in this study (89%). The most frequent mutation was an aberrant splicing site (655 A or C to G) in intron 2, in 30% of these chromosomes. Deletions were found in 20%, and large gene conversions in 13% of these alleles. This screening allowed the characterization of 18 out of 30 LO chromosomes, the most frequent mutation being Val281Leu (37%). Severe mutations were found, in heterozygosis, in one third of LO patients.

Detection of delta F508 cystic fibrosis mutation by polymerase chain reaction from old paraffin-embedded tissues: a retrospective autopsy study.

This study analyzes the usefulness of the polymerase chain reaction technique in the detection of delta F508 mutation in 11- to 25-yr-old formalin-fixed paraffin-embedded tissues obtained from the autopsy of 38 cystic fibrosis patients (nine with meconium ileus). Two different pairs of oligonucleotide primers were used: C16 B/C16 D that amplify 98 and/or 95 bp and FQ1/FQ2 that amplify 50 and/or 47 bp. After two independent rounds of polymerase chain reactions with the two sets of primers, amplification products were obtained in 67.5% of the cases when using C16 B/C16 D primers and in all cases when using FQ1/FQ2 primers. Fifty percent of the chromosomes analyzed in the 29 patients without meconium ileus had the delta F508 mutation, which was present in 13 heterozygous and 8 homozygous patients. The remaining eight cystic fibrosis patients did not carry that mutation. These results are similar to those reported in cystic fibrosis patients from Spain. In the meconium ileus group, we found a higher than expected proportion of delta F508 mutation; all patients showed the delta F508 mutation in at least one chromosome, seven patients (77.8%) being homozygous and two (22.2%) heterozygous. Present results indicate that delta F508 mutation can be detected by polymerase chain reaction in old paraffin-embedded tissues when appropriate primers are used.(ABSTRACT TRUNCATED AT 250 WORDS)

CF2603/4delT, a new frameshift mutation in exon 13 of the cystic fibrosis transmembrane conductance regulator (CFTR) gene.

By using temperature gradient gel electrophoresis to screen for mutations in cystic fibrosis (CF) patients, we have found a new mutation in the CF transmembrane conductance regulator gene. It is a frameshift mutation named CF2603/4delT located at the 3'-end of exon 13. A thymidine at position 2603 or 2604 is lost. The mutation eliminates an MseI site and, therefore, can be screened by restriction enzyme analysis.

[Evaluation of the Coulter STKS hematologic autoanalyzer]. / Valoración del autoanalizador hematológico Coulter STKS.

PURPOSE: To express the results of an evaluation of the Coulter STKS blood cell counter. MATERIAL AND METHODS: The following data were collected: inaccuracy, carry-over, linearity, comparison with other systems, suspect morphology alarms, stability of calibration, time-stability of samples and working velocity. The protocol recommended by the ICSH was followed for the first four of the above. RESULTS: Good results were achieved for cell counts and derived parameters. The leucocyte differential count showed fair results for neutrophils, lymphocytes, monocytes and eosinophils; false basophilia was usually associated with the presence of abnormal lymphocytes. No differences were found between adult and paediatric samples. The usefulness of the suspect morphology alarms was also evaluated; they were found fairly sensitive for the presence of stabs, large amounts of atypical lymphocytes and blast cells, and moderately sensitive for erythroblasts and platelet clumps. The predictive value of alarms was found high in any type of pathology, but scarcely valuable when considering the pathology indicated by the alarm, such predictive value being noticeably increased upon associated alarms. CONCLUSION: The Coulter STKS cell counter appears as adequate for the haematology laboratory running daily high number of samples with high pathologic rates.

Lipovitellin inhibition of Artemia trypsin-like proteinase: a role for a storage protein in regulating proteinase activity during development.

Artemia trypsin-like proteinase has been reported previously to be highly inhibited in the embryo (B. Ezquieta and C.G. Vallejo (1985) Comp. Biochem. Physiol. 82B, 731-736). We report now that Artemia lipovitellin, the major storage protein complex, inhibits the proteinase. We have carried out an in vitro study of the characteristics of the inhibition. Lipovitellin, a glycolipoprotein of high molecular mass (650 kDa), behaves initially as a substrate but after a limited proteolysis becomes an inhibitor of the proteinase. The enzyme although inhibited in the hydrolysis of the protein substrate retains activity toward low molecular weight substrates. The residual activity on the protein substrate is inhibited by small inhibitors of the proteinase. These features of lipovitellin inhibition are reminiscent of the trap mechanism of alpha 2-macroglobulin inhibition, previously proposed as suitable for regulating proteolytic processes involved in development. Inhibition by lipovitellin is greater at low temperatures and has been determined at 17 and 37 degrees C, in the lower and higher part of the viable temperature range of Artemia development. At high temperature the proteinase hydrolyzes the inhibitor quite efficiently and the inhibition is lower. The inhibition by lipovitellin appears specific for Artemia trypsin-like proteinase when compared with other control pairs protein/proteinase. The results may provide support for an additional role of storage proteins as developmental inhibitors of proteinases.