Kallmann syndrome: towards molecular pathogenesis.

Gonadotropin Releasing Hormone (GnRH) is a key regulator of reproduction and sexual behaviour. During the last decade, embryological studies have clarified the question of the early development of GnRH-synthesising neurones before the onset of neurosecretion. These studies have revealed the existence of a topographical link between GnRH-synthesising neurones and the embryonic olfactory system, thereby shedding new light on Kallmann syndrome, a developmental disease characterised by the association of hypogonadotropic hypogonadism and anosmia (or hyposmia). Although Kallmann syndrome was identified as an inherited disease in the forties, familial cases of the disease are infrequent. However, the identification, by positional cloning strategies, of the gene underlying the X-chromosome linked form of the disease (KAL-1) has opened the way to molecular pathophysiology. KAL-1 encodes an extracellular glycoprotein of compound modular structure. The protein, named anosmin-1, has been produced in a transfected mammalian cell line and purified. Polyclonal and monoclonal antibodies have been generated, which allowed us to study the distribution of the protein during the period of human organogenesis (4--10 embryonic weeks), by immunohistofluorescence. During this developmental period, anosmin-1 is a locally restricted component of various extracellular matrices (interstitial matrices and basement membranes). Later in embryonic life, KAL-1 expression apparently becomes restricted to definite neuronal populations. Based on the distribution of anosmin-1 in the early olfactory system, the pathogenesis of the olfactory loss and GnRH deficiency in X-linked Kallmann syndrome is discussed.

Chromosome abnormality in Kallmann syndrome.

We report on an individual with Kallman syndrome (KS) and a balanced de novo translocation (7;12)(q22,q24). None of 6 full sibs, 3 half-sibs, or parents have KS or this chromosome translocation. This is the only known report of KS with a chromosome abnormality. This may represent a spurious association or genetic heterogeneity vis-a-vis the reported linkage of KS to the steroid sulphatase gene on the X chromosome. The pathophysiology and genetics of KS are discussed.

The arrest of luteinizing hormone-releasing hormone neuronal migration in the genetic arhinencephalic mouse embryo (Pdn/Pdn).

From previous observations, it was suggested that non-attachment of the olfactory nerve to the telencephalon blocked the induction of the olfactory bulbs in genetic arhinencephalic mouse embryos (Pdn/Pdn). The olfactory nerve ends in a tangle beneath the forebrain in these embryos. From these observations, we speculated that the migration of luteinizing hormone-releasing hormone (LHRH) neurons might be disturbed in the olfactory nerve. A mass of LHRH neurons was observed in the end of the olfactory nerve fibers, but LHRH neurons were found in the hypothalamus in Pdn/Pdn embryos on day 16 of gestation. Narrow by-paths were found between the olfactory nerve and the forebrain, and the migration of LHRH neurons through these by-paths was observed in Pdn/Pdn embryos on day 13 of gestation. From the reports that a gene deleted in the arhinencephalic syndrome (Kallmann's syndrome) shares homology with neural cell adhesion molecules (N-CAM), it was speculated that non-attachment of the olfactory nerve in the Pdn/Pdn embryo might be associated with abnormalities of N-CAM. The axon fibers of the olfactory nerve reacted specifically with anti-N-CAM IgG both in +/- (+/+ and/or Pdn/+) and Pdn/Pdn on day 11.5 and 12, but not on day 13 and 16 of gestation. The axon fibers of the olfactory nerve were positive to anti-N-CAM IgG specifically just during the developmental period that the olfactory nerve fibers attached to the telencephalon. It is still not clear whether non-attachment of the olfactory nerve may be associated with N-CAM or not from the present observations.

Eyes in arhinencephalic syndromes.

The ocular features of eight cases of arhinencephaly have been described. Prediction of the degree of brain involvement from the eye defects could not be made, but eye abnormalities were present in all cases. The relationship of these syndromes to chromosomal abnormalities is emphasized. In the less severe cases treatable endocrine dysgenesis must be excluded.

Newborn with transverse facial cleft associated with polyhydramnios.

We describe here the case of a female newborn baby with a bilateral complete transverse facial cleft. Obstetrical ultrasound had revealed an increased amount of amniotic fluid from 28 weeks' gestation without fetal hydrops or congenital anomalies. A 1900-g baby girl born at 36 weeks' gestation presented with bilateral wide facial clefts with macrostomia, microphthalmia, nose, and auricular deformities. Her breathing was dependent on life support, which was discontinued 2 hours after birth. An autopsy revealed no congenital malformations in vital organs but the absence of the olfactory nerves. Polyhydramnios and respiratory arrest after birth were presumed to be due to central disintegration of swallowing and breathing, in this case with brain anomaly.

Disrupted compartmental organization of axons and dendrites within olfactory glomeruli of mice deficient in the olfactory cell adhesion molecule, OCAM.

There is an overall topographic connectivity in the axonal projections of olfactory sensory neurons from the olfactory epithelium (OE) to the olfactory bulb (OB). The molecular determinants of this overall topographic OE-OB connectivity are not known. For 20 years, the intriguing expression pattern of the olfactory cell adhesion molecule (OCAM) has made it the leading candidate as determinant of overall topographic OE-OB connectivity. Here, we have generated a strain of OCAM knockout mice by gene targeting. There were no obvious alterations in the distribution of olfactory sensory neurons within the OE or in the coalescence of axons into specific glomeruli. However, the compartmental organization of dendrites and axons within the glomeruli was disrupted. Surprisingly, the mutant mice exhibited an increase in olfactory acuity; they appeared to have a better sense of smell. Thus, despite its striking expression pattern, OCAM is not essential for overall topographic OE-OB connectivity. Instead, OCAM is required for establishing or maintaining the compartmental organization and the segregation of axodendritic and dendrodendritic synapses within glomeruli.

Hypotelorism in trisomy 1-producing mice.

Trisomy 1 embryos in mice are smaller in all dimensions, showing a developmental retardation as compared with euploid mice. Very rarely the trisomic embryos develop a typical hypotelorism with holoprosencephalon and missing olfactory nerves. Corneal distances, angles between the optic nerves and further microscopic examination showed no intermediate forms between the trisomy 1 embryos and the rare trisomy 1 embryo with hypotelorism. There seems to be a threshold, beyond which the major developmental derangement occurred. This is an experimental model showing parallels to the occasional varying phenotypes in human trisomies.

Characterization of olfactory nerve abnormalities in Twirler mice.

The sense of smell is perceived by olfactory receptor neurons (ORN) present in the olfactory epithelium located in the posterosuperior aspect of the nasal cavity. The axons of these ORN migrate to the olfactory bulb (OB), forming a nervous layer on the outermost part of the bulb, and finally synapse in glomerular structures in the OB. The ORN are unique in that they are constantly being renewed throughout life. We characterized the defects in the nasal cavity and olfactory nervous supply of Twirler (Tw) mice by histological and immunohistochemical means. Tw homozygotes have previously been shown to present with midfacial abnormalities in the form of clefts of the lip and palate (Lyon, 1958; Gong et al., 2000). We found that in the Tw homozygotes, the OB was abnormally shaped, the skeletal framework underlying the OB was disrupted, and the morphology of the nasal cavity was altered with poorly defined nasal turbinates. Immunohistochemical staining with antibodies that marked nerves in general (PGP 9.5) and mature ORN (omp) in the olfactory epithelium at two different embryonic stages and in newborn mice revealed the stratification of the olfactory epithelium in Tw homozygotes, albeit slightly thinner compared to wildtype. A striking difference in the olfactory epithelium was the lack of differentiation of the ORN in Tw homozygotes and the reduced axonal input to the OB. In Tw homozygotes at 14.5 days of embryonic development, the presence of many mature ORN found randomly in the mesenchyme suggests the loss of olfactory pathfinding cues to the OB. It is believed that the lack of appropriate pathfinding cues observed in the Tw homozygotes was responsible for the OB not having the appropriate trophic effect on the development and maturation of the ORN as had been observed in partially bulbectomized animals. The defects in the Twirler may prove to be a valuable system to analyze problems in olfactory pathfinding and maturation.

A molecular approach to the pathophysiology of the X chromosome-linked Kallmann's syndrome.

The human KAL gene is responsible for the X chromosome-linked Kallmann's syndrome, which consists of an association between hypogonadotropic hypogonadism and anosmia (or hyposmia). Additional symptoms are occasionally observed. The olfactory defect is associated with hypoplasia of the olfactory bulbs and tracts. The hypogonadism may be due to a defect in the embryonic migratory process of GnRH-synthesizing neurones from the olfactory pits up to the brain. The human and chicken KAL genes have been isolated. From the amino acid sequences deduced, it has been postulated that the KAL protein is an extracellular matrix component, with putative antiprotease activity and adhesion function. Various point mutations and, in a few cases, deletions of KAL have been detected in patients. By in situ hybridization, KAL expression has been studied during embryonic development in the chick. From embryonic day 2 (ED2) to ED8, the KAL gene is expressed in various endodermal, mesodermal and ectodermal derivatives, whereas the expression from ED8 is almost entirely restricted to definite neuronal populations in the central nervous system, most of which still express the gene after hatching. According to such a spatiotemporal pattern of expression, we suggest that the KAL gene is involved both in morphogenetic events and in late neuronal differentiation and/or neuronal trophicity. With respect to the olfactory system, the KAL gene is expressed in the mitral cells of the olfactory bulbs from ED8 onwards. In contrast, no expression of the KAL gene is detected at any stage in either the embryonic olfactory epithelium or the surrounding nasal mesenchyme. Therefore, assuming that similar conditions are found in the human embryo, we suggest that the olfactory anomaly in X-linked Kallmann's syndrome results from a central target cell defect. Current hypotheses regarding the pathophysiology of the GnRH deficiency are also discussed. In situ hybridization experiments in the human embryo, as well as characterization of the KAL protein, are in progress.

Development of the olfactory nerve: its relationship to the craniofacies.

Although absence of the olfactory bulbs is a relatively common occurrence seen in holoprosencephaly, in Kallman syndrome, and in a number of malformation syndromes, the extent to which it determines olfactory nerve development, as well as the part it plays in the morphogenesis of the nasal structures, is unknown. Cases of arhinencephaly ascertained at autopsy were studied in an effort to better understand the relationships between the olfactory nerve, bulb, and facies. Based on these studies, it is concluded that both olfactory receptor cells and olfactory nerves are present in arhinencephaly, that the olfactory nerves did not make contact with the brain in these cases, that the presence of olfactory nerves is independent of the severity of the central nervous system malformation, and that the shape of the nasal structures is not dependent on the presence of the olfactory nerve.

Prenatal diagnosis and investigation of a fetus with chondrodysplasia punctata, ichthyosis, and Kallmann syndrome due to an Xp deletion.

We report the prenatal diagnosis of a male fetus with X-linked recessive chondrodysplasia punctata (CDPX), steroid sulphatase (STS) deficiency, X-linked Kallmann syndrome (KAL), and a chromosome deletion at Xp22.31. Biochemical analysis of bone from this case indicates that CDPX is not a defect of vitamin K metabolism. Immunocytochemical study of the brain suggests that KAL is a defect in neuronal migration.