Juan Carlos Zenteno

Molecular analysis in true hermaphroditism: demonstration of low-level hidden mosaicism for Y-derived sequences in 46,XX cases

Abstract. True hermaphroditism (TH) is an unusual form of sex reversal, characterized by the development of testicular and ovarian tissue in the same subject. Approximately 60% of the patients have a 46,XX karyotype, 33% are mosaics with a second cell line containing a Y chromosome, while the remaining 7% are 46,XY. Molecular analyses have demonstrated that SRY is present in only 10% of TH with a 46,XX karyotype; therefore, in the remaining 90%, mutations at unknown X-linked or autosomal sex determining loci have been proposed as factors responsible for testicular development. True hermaphroditism presents considerable genetic heterogeneity with several molecular anomalies leading to the dual gonadal development as SRY point mutations or SRY hidden gonadal mosaicism. In order to identify genetic defects associated with subjects with the disease, we performed molecular analyses of the SRY gene in DNA from blood leukocytes and gonadal tissue in 12 true hermaphrodites with different karyotypes. Our results using PCR and FISH analyses reveal the presence of hidden mosaicism for SRY or other Y sequences in some patients with XX true hermaphroditism and confirms that mosaicism for SRY limited to the gonads is an alternative mechanism for testicular development in 46,XX true hermaphrodites.

Two SRY-negative XX male brothers without genital ambiguity

Abstract We report a Mexican family in which two sibs were identified as “classic” XX males without genital ambiguities. Molecular studies revealed that both patients were negative for several Y sequences, including SRY. A review of familial cases disclosed that this is the first family where a complete male phenotype was observed in Y-negative XX male non-twin brothers. These data suggest that an inherited loss-of-function mutation, in a gene participating in the sex-determining cascade, can induce normal male sexual differentiation in the absence of SRY.

Molecular analysis of the anti-Müllerian hormone, the anti-Müllerian hormone receptor, and galactose-1-phosphate uridyl transferase genes in patients with the Mayer-Rokitansky-Küster-Hauser syndrome

Introduction.Müllerian agenesis, also named the Mayer-Rokitansky-Küster-Hauser syndrome (MRKHS) or vaginal aplasia, is the second most common cause of primary amenorrhea. It is characterized by the congenital absence of the Müllerian structures including the Fallopian tubes, the uterus, and the internal portion of the vagina in an otherwise normally feminized 46,XX subject. Most cases are sporadic in inheritance, but the occurrence of some patients with chromosomal translocations or even familial aggregates suggest a genetic basis for the disease, although the etiology of the disease is still unknown. It has been suggested that activating mutations in the anti-Müllerian hormone (AMH) or in its receptor (AMHRII) are potential sources for the defect.Methods.In this study we describe the molecular analysis of both the AMH and AMHR genes in a group of 15 patients with Müllerian agenesis. After sequencing all exons and exon/intron junctions of both genes, we were not able to detect any deleterious mutation.Results.Five new polymorphisms, 2 of them in the AMHRII gene and 3 of them in the AMH gene, were identified. No significant differences between patients and controls were observed in the frequency of a given polymorphism.Conclusion.This work reinforces the view that molecular defects in the AMH or AMHR are unlikely sources for the MRKHS syndrome.

Contiguous gene syndrome due to deletion of the first three exons of the Kallmann gene and complete deletion of the steroid sulphatase gene

Large terminal or interstitial deletions of the 22.3 region on the short arm of the X chromosome cause contiguous gene syndromes. Kallmann syndrome (hypogonadotrophic hypogonadism with anosmia or hyposmia) associated with X‐linked ichthyosis, due to a contiguous gene syndrome, is an uncommon finding. Genetic defects have been demonstrated in the Xp22.3 region, explaining the presence of one or both entities in affected individuals. In this report we describe the molecular findings of a patient with Kallmann syndrome and X‐linked ichthyosis.

Phenotypic heterogeneity associated with identical mutations in residue 870 of the androgen receptor.

Background/Aims: Mutations in the androgen receptor (AR) gene result in an X-linked recessive form of male pseudohermaphroditism known as the androgen-insensitivity syndrome (AIS). The alterations most frequently observed are missense or nonsense point mutations in exons 4–8 of the AR gene that affect the steroid-binding domain of the receptor in subjects with various degrees of androgen resistance. Despite the increasing number of AR mutations identified, a reliable genotype-phenotype correlation has not been established and individuals with the same molecular defect may exhibit different phenotypes. Here, we studied a patients with an AIS characterized by bilateral gynecomastia, normal male external genitalia, and normal sperm counts. Methods: Exon-specific polymerase chain reaction, single-stranded conformational polymorphism, and sequencing analysis of the subject’s AR gene were performed in addition to hormone-binding assays in skin fibroblasts from the patient. Results: A point mutation at codon 870 of the AR, changing alanine to valine, was detected. Conclusion: As AR missense mutations changing alanine 870 to valine have been previously described in 3 unrelated patients showing severe AIS phenotypes, we conclude that phenotypic heterogeneity associated to identical mutations in the AR gene is probably due to individual functional differences in AR coregulator molecules.

A novel filamin A D203Y mutation in a female patient with otopalatodigital type 1 syndrome and extremely skewed X chromosome inactivation

Otopalatodigital syndrome type 1 (OPD1) [OMIM 311300] is an X‐linked dominant multiple congenital anomalies disease mainly characterized by a generalized skeletal dysplasia, mild mental retardation, hearing loss, cleft palate, and typical facial anomalies. OPD1 belongs to a group of X‐linked skeletal dysplasias known as oto‐palato‐digital syndrome spectrum disorders that also include OPD2, Melnick–Needles syndrome (MNS), and frontometaphyseal dysplasia (FMD). Recently, it has been demonstrated that mutations in the gene encoding the cytoskeletal protein Filamin A (FLNA) are responsible for this group of clinically overlapping human syndromes. We present the phenotypic and molecular data of a sporadic female patient clinically diagnosed with an OPD1 syndrome who carried a novel FLNA point mutation resulting in an Asp203Tyr substitution in the actin‐binding domain of the protein. X‐inactivation analyses demonstrated an extremely skewed pattern towards her maternal chromosome. Our results add to the molecular spectrum of the oto‐palato‐digital related syndromes and contribute to the delineation of phenotype‐genotype correlation in this group of X‐linked skeletal disorders.

An atypical contiguous gene syndrome: molecular studies in a family with X–linked Kallmann's syndrome and X‐linked ichthyosis

Kallmanns syndrome (KS) is characterized by hypogonadotrophic hypogonadism in association with anosmia or hyposmia. This entity can be associated with X‐linked ichthyosis (XLI) in a contiguous gene syndrome. Genetic defects have been demonstrated on the Xp22.3 region explaining the presence of one or both entities in affected individuals. In this report we describe the molecular findings in four patients, pertaining to a three generation family, with KS which was associated with XLI in two of them.

Clinical expression and SRY gene analysis in XY subjects lacking gonadal tissue.

In several syndromes genetic males lack gonadal tissue. A range of phenotypes are seen, which varies from complete female external genitalia to anorchic subjects with sexual infantilism. Differences in phenotypic expression depend on the stage at which testes degenerated during intrauterine development. Although most cases of these syndromes are sporadic, several instances of familial recurrence suggest a genetic origin. To help elucidate the source, we performed molecular analysis of the complete SRY gene open reading frame in two subjects with true agonadism and in two with anorchia. Our results add to previous findings indicating that molecular defects in SRY are not readily identified as a cause of these syndromes.

A de novo phe67leu mutation in the SRY gene in a patient with complete 46,XY gonadal dysgenesis

The sex-determining region of the Y chromosome (SRY) gene initiates the process of male sex differentiation in mammalians. In humans, mutations in the SRY gene have been reported to account for 10-15% of the XY sex reversal cases. In this report we describe the clinical, endocrinological and molecular data of a patient with complete 46,XY gonadal dysgenesis caused by a de novo mutation affecting SRY amino acid phenylalanine at position 67 (F67L), located within the highly conserved high mobility group (HMG) box coding region of the gene.

A de novo heterozygous point mutation in the p63 gene causing the syndrome of ectrodactyly, ectodermal dysplasia and facial clefting.

with weak expression of fibronectin also in the lower dermis. In contrast, vitronectin and undulin were not expressed in basement membranes but instead only in interstitial dermal connective tissue, with weaker expression of undulin in the lower dermis (see also Fig. 1a). Expression of collagen IV and laminin was restricted to basement membranes. In most cases and with most ECM proteins studied, there was a striking modulation of expression in the three inflammatory skin diseases which was, however, not consistent in all specimens (Table 2). In psoriasis, most ECM proteins were downregulated, except for an upregulation of tenascin in about 70% of the specimens and of undulin subepidermally (Fig. 1b). In lichen planus and morphoea, fibronectin and tenascin were upregulated in the dermis, whereas vitronectin and undulin were unchanged. As in psoriasis, laminin and collagen IV were decreased and ⁄ or fragmented in almost all cases at the dermoepidermal junction, whereas expression at basement membranes around vessels and appendages was unaltered in comparison with normal skin (Fig. 2). A close apposition to ECM molecules was seen only for laminin and collagen IV at both the dermoepidermal junction and around vessels (not shown). The present findings are novel in that, for the first time, a close apposition of mature mast cells to the major basement components collagen IV and laminin, particularly around vascular basement membranes, is demonstrated in normal and diseased skin. Furthermore, our findings fit previous in vitro studies where collagen IV and laminin have been shown to interact with immature murine and leukaemic human mast cells (HMC-1 cell line). The lack of contacts with fibronectin and vitronectin, as described before, may be due to differences in cultured immature vs. normal tissue mast cells studied here. The present data are in agreement with previous studies regarding the distribution and expression of ECM proteins in normal and diseased skin (for example, see Schalkwijk et al.). The findings regarding tenascin in cutaneous lichen planus, vitronectin in morphoea, and undulin in lichen planus and morphoea are, however, new. The functional significance of the present findings most probably relates to the interaction of mast cells with basement membranes and the effect of these cells on keratinocytes, endothelial cells and fibroblasts, by the possible induction of production of a number of growth factors for these cells. This should allow for the maintenance of the functional integrity of vessels and epithelia via the stimulation of laminin and collagen IV production.