Ignacio Bergadá

Mutations in the PCNA-binding domain of CDKN1C cause IMAGe syndrome

IMAGe syndrome (intrauterine growth restriction, metaphyseal dysplasia, adrenal hypoplasia congenita and genital anomalies) is an undergrowth developmental disorder with life-threatening consequences. An identity-by-descent analysis in a family with IMAGe syndrome identified a 17.2-Mb locus on chromosome 11p15 that segregated in the affected family members. Targeted exon array capture of the disease locus, followed by high-throughput genomic sequencing and validation by dideoxy sequencing, identified missense mutations in the imprinted gene CDKN1C (also known as P57KIP2) in two familial and four unrelated patients. A familial analysis showed an imprinted mode of inheritance in which only maternal transmission of the mutation resulted in IMAGe syndrome. CDKN1C inhibits cell-cycle progression, and we found that targeted expression of IMAGe-associated CDKN1C mutations in Drosophila caused severe eye growth defects compared to wild-type CDKN1C, suggesting a gain-of-function mechanism. All IMAGe-associated mutations clustered in the PCNA-binding domain of CDKN1C and resulted in loss of PCNA binding, distinguishing them from the mutations of CDKN1C that cause Beckwith-Wiedemann syndrome, an overgrowth syndrome.

Establishment of testicular endocrine function impairment during childhood and puberty in boys with Klinefelter syndrome.

Objective  To precisely characterize the chronology of testicular endocrine function impairment during childhood and adolescence in patients with Klinefelter syndrome.

Male hypogonadism: an extended classification based on a developmental, endocrine physiology‐based approach

Normal testicular physiology results from the integrated function of the tubular and interstitial compartments. Serum markers of interstitial tissue function are testosterone and insulin‐like factor 3 (INSL3), whereas tubular function can be assessed by sperm count, morphology and motility, and serum anti‐Müllerian hormone (AMH) and inhibin B. The classical definition of male hypogonadism refers to testicular failure associated with androgen deficiency, without considering potential deficiencies in germ and Sertoli cells. Furthermore, the classical definition does not consider the fact that low basal serum testosterone cannot be equated to hypogonadism in childhood, because Leydig cells are normally quiescent. A broader clinical definition of hypogonadism that could be applied to male patients in different periods of life requires a comprehensive consideration of the physiology of the hypothalamic‐pituitary‐testicular axis and its disturbances along development. Here we propose an extended classification of male hypogonadism based on the pathophysiology of the hypothalamic‐pituitary‐testicular axis in different periods of life. The clinical and biochemical features of male hypogonadism vary according to the following: (i) the level of the hypothalamic‐pituitary‐testicular axis primarily affected: central, primary or combined; (ii) the testicular cell population initially impaired: whole testis dysfunction or dissociated testicular dysfunction, and: (iii) the period of life when the gonadal function begins to fail: foetal‐onset or postnatal‐onset. The evaluation of basal testicular function in infancy and childhood relies mainly on the assessment of Sertoli cell markers (AMH and inhibin B). Hypergonadotropism should not be considered a sine qua non condition for the diagnosis of primary hypogonadism in childhood. Finally, the lack of elevation of gonadotropins in adolescents or adults with primary gonadal failure is indicative of a combined hypogonadism involving the gonads and the hypothalamic‐pituitary axis.

Sertoli cell proliferations of the infantile testis: an intratubular form of Sertoli cell tumor?

We report on six boys with intratubular Sertoli cell proliferations (ISCPs), studied by routine histologic methods, electron microscopy, and immunohistochemistry of anti-müllerian hormone (AMH), inhibin &agr;-subunit, 3&bgr;-hydroxysteroid dehydrogenase (3&bgr;-HSD), proliferative cellular nuclear antigen, and p53, and carefully followed for extended periods with periodic clinical examinations, testicular ultrasonographies, and determinations of serum levels of AMH and inhibin B. Peutz–Jeghers syndrome was found in four of six patients, and gynecomastia occurred in five of six patients. One boy had isosexual pseudoprecocity. ISCPs were observed as multiple foci of seminiferous tubules with large and proliferated Sertoli cells replacing germ cells and limited by the basement membrane. Mitotic figures, atypia, and/or interstitial invasion were not observed. Bilateral ISCPs were the only pathologic finding in three patients (patient nos. 1–3) and were associated with a microscopic tumor that resembled a large-cell calcifying Sertoli cell tumor (LCCSCT) in a fourth patient (patient no. 4). In the two remaining patients (patient nos. 5 and 6) ISCPs and LCCSCT were found in both testes. Ultrastructural examination showed large Sertoli cells, with round nuclei, sparse organelles, and some glycogen. Inhibin &agr;-subunit immunolocalization was positive in the five patients in whom it was determined (patient nos. 2–6), AMH was positive in those ISCPs associated with tumors (patient nos. 4–6) and negative in isolated ISCPs (patient nos. 2 and 3); 3&bgr;-HSD and PCNA were variable, and p53 was negative in all ISCPs. Patient nos. 1–4 have been followed for 2–19 years. One of them is currently entering puberty, the other two have already completed puberty and have testes of normal size, and the remaining one is an adult with clinically normal testes and sperm production. None of these patients had evidence of tumor development during follow-up as shown by serial ultrasonographies and serum levels of AMH and inhibin B. Patient nos. 5 and 6 who had bilateral ISCPs and LCCSCT were orchidectomized and evolved for 2–10 years after surgery without tumor recurrence. The prognostic significance of ISCPs, particularly when they are the only pathologic finding in a testicular biopsy, is a matter of controversy. Based on the long normal evolution, we recommend a conservative approach to therapy. The bilateral and multicentric character of ISCPs and their association with Sertoli tumors and Peutz–Jeghers syndrome suggest that they represent either proliferative lesions with tumorigenic potential or the intraepithelial stage in the evolution of some testicular Sertoli cell tumors.

Mutations of the steroid 21-hydroxylase gene in an Argentinian population of 36 patients with classical congenital adrenal hyperplasia.

In several studies carried out in USA and Europe, gene deletions, large gene conversions and six point mutations accounted for over 90% of the mutated alleles reported in classical congenital hyperplasia (CAH). In order to know the relative frequencies of mutations in a Latin-American population, the CYP21 active gene was analyzed in 42 patients with CAH belonging to 36 families attending two Argentinian clinics. The salt wasting form was diagnosed in 24 index cases and the simple virilized form in 12. When available, parents were also studied. DNA was extracted from peripheral blood leukocytes and specific PCR amplification of four different fragments of the CYP21 gene was carried out, followed by electrophoresis of the amplified product. The four fragments include segments of the gene containing the six most frequently reported abnormalities in classical CAH: IN2, EX3, R356W, cluster EX6 and I172N. Point mutations were studied by allelic specific oligonucleotide hybridization; Q318X was studied by digestion of the PCR product with PsT1 restriction enzyme and electrophoresis on 6% non-denaturing polyacrylamide gels. Deletions and macroconversions as well as confirmation of homozygote point mutations were studied by Southern blotting. Percentage distribution of abnormalities was as follows: deletion/macroconversion 18, IN2 18, I172N 15.3, Q318X 13.8, R356W 5.5, EX3 2.7, cluster EX6 0, not characterized 26.7. The complete genotype could be determined in 20 families while in 12 additional ones, the mutation was detected in one allele. Deletion/macroconversion, IN2, EX3 and Q318X were detected more frequently in salt wasting patients while I172N and R356W were found in simple virilized patients. However, genotype was not always concordant with phenotype. It is concluded that there are differences in the frequency of several gene mutations and in that of deletion/macroconversion between this Latin-American population and several reported American and European populations. In particular the percentage of deletion/macroconversion, IN2, EX3 and cluster EX6 was lower while I172N was higher in our Latin-American population. Furthermore the frequency of mutations not characterized was larger. This information is useful to delineate appropriate strategies for prenatal diagnosis in this particular population.

Steroid 21‐hydroxylase gene mutational spectrum in 454 Argentinean patients: genotype–phenotype correlation in a large cohort of patients with congenital adrenal hyperplasia

Objective  To report genotype–phenotype correlation in a large cohort of patients.

Latin American Consensus: Children Born Small for Gestational Age

BackgroundChildren born small for gestational age (SGA) experience higher rates of morbidity and mortality than those born appropriate for gestational age. In Latin America, identification and optimal management of children born SGA is a critical issue. Leading experts in pediatric endocrinology throughout Latin America established working groups in order to discuss key challenges regarding the evaluation and management of children born SGA and ultimately develop a consensus statement.DiscussionSGA is defined as a birth weight and/or birth length greater than 2 standard deviations (SD) below the population reference mean for gestational age. SGA refers to body size and implies length-weight reference data in a geographical population whose ethnicity is known and specific to this group. Ideally, each country/region within Latin America should establish its own standards and make relevant updates. SGA children should be evaluated with standardized measures by trained personnel every 3 months during year 1 and every 6 months during year 2. Those without catch-up growth within the first 6 months of life need further evaluation, as do children whose weight is ≤ -2 SD at age 2 years. Growth hormone treatment can begin in SGA children > 2 years with short stature (< -2.0 SD) and a growth velocity < 25th percentile for their age, and should continue until final height (a growth velocity below 2 cm/year or a bone age of > 14 years for girls and > 16 years for boys) is reached. Blood glucose, thyroid function, HbA1c, and insulin-like growth factor-1 (IGF-1) should be monitored once a year. Monitoring insulin changes from baseline and surrogates of insulin sensitivity is essential. Reduced fetal growth followed by excessive postnatal catch-up in height, and particularly in weight, should be closely monitored. In both sexes, gonadal function should be monitored especially during puberty.SummaryChildren born SGA should be carefully followed by a multidisciplinary group that includes perinatologists, pediatricians, nutritionists, and pediatric endocrinologists since 10% to 15% will continue to have weight and height deficiency through development and may benefit from growth hormone treatment. Standards/guidelines should be developed on a country/region basis throughout Latin America.

Role of inhibins in childhood and puberty.

Inhibins, produced mainly in the gonads, suppress FSH synthesis. The bioactive dimeric forms of inhibin (A and B) have been proposed as peripheral markers of Sertoli and granulosa cell function. The determination of serum dimeric inhibins from birth through adulthood reflects a distinct pattern of both inhibins in males and females. Concomitantly with the gonadotrophin surge, an important production of inhibin B is observed during the first months of life. In males, inhibin B levels are higher than in females and persist elevated up to childhood, whereas in females they decrease up to prepubertal levels by 6 months of age. In girls, high serum levels of inhibin A are observed during the first two months of life; thereafter, they are undetectable until puberty. An active secretion of inhibin B persists in both males and females in the period of maximal LHRH pulse generator restraint; however, the possible gonadotrophin dependence of this production remains controversial. At puberty, a progressive rise in serum inhibin B occurs concomitantly with the increased production of sex steroids in both males and females. A similar secretion pattern of inhibin A is observed in girls. This increment is mainly exerted by gonadotrophins and modulated by multiple paracrine/autocrine mechanisms within the ovary and the testis that regulate the dimerization of the inhibin subunits throughout pubertal maturation. The differences observed in males and females between circulating dimeric inhibins in relation to gonadotrophins and sex steroid concentrations from birth through puberty has opened a new perspective for research in human reproduction. These new markers may contribute to a better knowledge of the regulation of the hypothalamic-pituitary-gonadal axis function and the physiopathology of the mechanisms involved in sexual differentiation and/or fertility disorders.

CDKN1C mutations: two sides of the same coin

Cyclin-dependent kinase (CDK)-inhibitor 1C (CDKN1C) negatively regulates cellular proliferation and it has been shown that loss-of-function mutations in the imprinted CDKN1C gene (11p15.5) are associated with the overgrowth disorder Beckwith-Wiedemann syndrome (BWS). With recent reports of gain-of-function mutations of the PCNA domain of CDKN1C in growth-retarded patients with IMAGe syndrome or Silver-Russell syndrome (SRS), its key role for growth has been confirmed. Thereby, the last gap in the spectrum of molecular alterations in 11p15.5 in growth-retardation and overgrowth syndromes could be closed. Recent functional studies explain the strict association of CDKN1C mutations with clinically opposite phenotypes and thereby contribute to our understanding of the function and regulation of the gene in particular and epigenetic regulation in general.

Altered serum profile of inhibin B, Pro‐αC and anti‐Müllerian hormone in prepubertal and pubertal boys with varicocele

objective  Anti‐Müllerian hormone (AMH) and inhibin B are reliable markers of Sertoli cell function. The aim of the present study was to assess the functional state of Sertoli cells in order to detect early changes in the testicular function of prepubertal and pubertal patients with untreated grade II or III varicocele.