KAL1 Research Articles

Characterization of the two zebrafish orthologues of the KAL-1 gene underlying X chromosome-linked Kallmann syndrome

The gene underlying X chromosome-linked Kallmann syndrome, KAL-1, has been identified for several years, yet its role in development is still poorly understood. In order to take advantage of the zebrafish as a model in developmental genetics, we isolated the two KAL-1 orthologues, kal1.1 and kal1.2, in this species. Comparison of deduced protein sequences with the human one shows 75.5 and 66.5% overall homology, respectively. The most conserved domains are the whey acidic protein-like domain and the first of four fibronectin-like type III repeats. However, kal1.2 putative protein lacks the basic C-terminal domain (20 residues) found in kal1.1 and KAL-1. The expressions of kal1.1 and kal1.2 were studied in the embryo between 6 and 96 hours post fertilization using whole-mount in situ hybridization. Although a few structures express both genes, kal1.1 and kal1.2 expression patterns are largely non-overlapping. Taken together, these patterns match fairly well those previously reported for human KAL-1 and chicken kal1. As regards the olfactory system, kal1.1 is expressed, from 37 h.p.f. onward, in the presumptive olfactory bulbs, whereas kal1.2 transcript is only detected, from 48 h.p.f., in the epithelium of the nasal cavity. The relevance of the zebrafish as an animal model for studying both the function of KAL-1 in normal development and the developmental failure leading to the olfactory defect in Kallmann syndrome, is discussed.

The Molecular Basis of Human Hypogonadotropic Hypogonadism

Patients with hypogonadotropic hypogonadism (HH) present with delayed puberty, infertility, and low serum gonadotropins. The molecular basis for most cases of HH is unknown, but single gene mutations have been described for some hypothalamic and pituitary genes. Kallmann syndrome due to KAL gene mutations and adrenal hypoplasia congenita/HH caused by AHC gene mutations are both X-linked recessive disorders. Mutations in the gonadotropin releasing hormone receptor, leptin, and the leptin receptor cause autosomal recessive HH. In addition, isolated deficiencies of follicle stimulating hormone and luteinizing hormone in the corresponding specific β-subunit genes and PROP1 gene mutations represent pituitary deficiency states, resulting in a phenotype of HH. Despite these remarkable advances in our understanding of human HH, the cause of approximately 90% remains unknown.

Neue Erkenntnisse zur Genetik des Kallmann-Syndroms und des Idiopathischen Hypogonadotropen Hypogonadismus (IHH)*

Kallmann syndrome (KS) is characterised by hypogonadotropic hypogonadism due to GnRH deficiency and concomitant anosmia. The latter is absent in idiopathic hypogonadotropic hypogonadism (IHH), an entity otherwise indistinguishable from KS. Most patients with KS or IHH have no positive family history. Some of the familial cases of KS follow an X-linked recessive pattern of inheritance. This subtype is caused by deletions or point mutations in the KAL-1 gene. Its protein product, anosmin-1, is believed to play an important role in the morphogenesis of the peripheral olfactory system. Recent studies have shown that only a minority of patients with Kallmann syndrome carry mutations in the KAL-1 gene. Kallmann syndrome as well as IHH may also follow an autosomal dominant or autosomal recessive pattern of inheritance. With regard to the latter case, a single family has been described with mutations in the GnRH receptor gene. Other genes causing the autosomal subforms of KS/IHH remain to be identified. In sibships with multiple affected members marked clinical variability has been observed. Within a single family patients with either Kallmann syndrome, IHH, isolated anosmia, or only delayed or incomplete pubertal development may be seen. This hints at a close nosological relationship between these clinical phenotypes which may share a common - so far however rarely identifiable - genetic basis.

Renal abnormalities in patients with Kallmann syndrome.

To report experience in patients with Kallmann syndrome (KS) in whom urography was used to establish the type and frequency of renal anomalies associated with the disorder. Of 19 patients with KS, 15 had the X-linked recessive form of the disease, whereas the remaining four were sporadic. Each patient underwent intravenous pyelography (IVP) using a non-ionic, low osmolarity contrast medium. Of the 19 patients with KS, 10 had kidney abnormalities; four presented with unilateral renal agenesis and six had less severe forms of renal abnormality (renal malrotation in four and bilateral dilatation of the calyces and pelves in two). One of the patients with unilateral renal agenesis carried a deletion in KAL, the gene responsible for the X-linked type of KS. Three of the four patients with renal malrotation had a confirmed X-linked recessive form and one carried a point mutation in KAL. These results suggest that kidney abnormalities are more frequent and diverse in patients with KS than previously reported. They also indicate that defects in the KAL gene may contribute to abnormal renal development. However, a review of the literature revealed no close correlation between KAL mutations and kidney anomalies in the X-linked type of disease. Taken together, these data suggest that KAL mutations are not invariably associated with failure of renal development and that additional factors (epigenetic or local) may compensate for defects in the KAL protein.

A Novel Aminoterminal Mutation in the KAL-1 Gene in a Large Pedigree with X-Linked Kallmann Syndrome

Kallmann syndrome is characterized by hypogonadotropic hypogonadism and anosmia. Autosomal dominant, autosomal recessive, and X-linked patterns of transmission have been described. The X-linked form of Kallmann syndrome (XLKS) is the least common of the three modes of inheritance and is caused by mutations in the putative cell adhesion protein, KAL-1. In a large pedigree with XLKS, direct sequencing of the KAL-1 gene revealed a duplication of 11 base pairs in exon 1, resulting in a frameshift and a premature stop at codon 34 of the 680 amino acid protein. The clinical features of the affected individuals in this pedigree provide further evidence in support of the idea that XLKS is associated with neurologic features that are not seen in other forms of the syndrome.

A recurrent missense mutation in the KAL gene in patients with X-linked Kallmann's syndrome.

Kallmann's syndrome (KS) is defined by the association of hypogonadotropic hypogonadism and anosmia or hyposmia. Segregation analysis in familial cases has demonstrated diverse inheritance patterns, suggesting the existence of several genes regulating GnRH secretion. Genetic defects have been demonstrated in the KAL gene, located on the Xp22.3 region, explaining the X-linked form of the disease. We report molecular findings regarding the KAL gene in 12 unrelated males with X-linked KS. PCR of the 14 exons of the KAL gene was performed on genomic DNA. PCR products of all exons were purified and sequenced. Genetic defects in the KAL gene were found in 7 patients. One exhibits a deletion from exon 3 to exon 5. Six individuals present a previously unidentified missense mutation in exon 11, consisting of a G to A substitution at codon 514 (GAA to AAA). In the remaining 5 individuals, no mutations were observed. We also found three different polymorphic changes. The first one, in exon 2, had not been reported previously. The other two were located at exons 11 and 12. The deletion described, comprises only part (exon 5) of the coding region of the first fibronectin type III-like repeat of the KAL protein. The rest of the deletion comprises part of the conserved cysteine-rich N-terminal region that corresponds to the whey acidic protein motif. The same missense mutation was found in 6 of the 12 patients, indicating the possibility that it derived from a common ancestor or suggesting the presence of a hot spot in this region of the gene.

A Recurrent Missense Mutation in the KAL Gene in Patients with X-Linked Kallmann's Syndrome

A Recurrent Missense Mutation in the KAL Gene in Patients with X-Linked Kallmann's Syndrome

The oral-facial-digital syndrome type 1 (OFD1), a cause of polycystic kidney disease and associated malformations, maps to Xp22.2-Xp22.3

Key features of the oral-facial-digital syndrome type 1 (OFD1) include malformations of the face, oral cavity and digits. In addition, the clinical phenotype often includes mental retardation and renal functional impairment. Approximately 75% of cases of OFD1 are sporadic, and the condition occurs almost exclusively in females. In familial cases, the most likely mode of inheritance is considered to be X-linked dominant with prenatal lethality in affected males. Therefore, the OFD1 gene product appears to have widespread importance in organogenesis and is essential for fetal survival. We have studied two kindreds in which the clinical course was dominated by polycystic kidney disease requiring dialysis and transplantation. Using polymorphic chromosome markers spaced at approximately 10 cM intervals along the X chromosome, we mapped the disease to a region on the short arm of the X chromosome (Xp22.2-Xp22.3) spanning 19.8 cM and flanked by crossovers with the markers DXS996 and DX7S105. There was a maximum lod score of 3.32 in an 'affecteds only' analysis using a marker within the KAL gene (theta = 0.0 ), thereby confirming the location of the gene for OFD1 on the X chromosome. The remainder of the X chromosome was excluded by recombinants in affected individuals. The importance of our findings includes the definitive assignment of this male-lethal disease to the X chromosome and the mapping of a further locus for a human polycystic kidney disease. Furthermore, this mapping study suggests a possible mouse model for OFD1 as the X-linked dominant Xpl mutant, in which polydactyly and renal cystic disease occurs, maps to the homologous region of the mouse X chromosome.

Genetic heterogeneity evidenced by low incidence of KAL-1 gene mutations in sporadic cases of gonadotropin-releasing hormone deficiency.

Isolated GnRH deficiency is a heritable condition characterized by a functional deficit in GnRH secretion. Familial cases with different modes of inheritance have been described, and the gene responsible for the X-linked form (KAL-1) has been identified. However, sporadic cases with no documented family history of GnRH deficiency account for the majority of the affected patients. For this reason, we sought to determine the frequency with which KAL-1 gene mutations occur in patients with sporadic GnRH deficiency. Only 1 of 21 patients with sporadic GnRH deficiency was found to bear a defect in the KAL-1 gene (a deletion of 14 bases starting at codon 464). Three types of polymorphic single base substitutions with no apparent correlation with GnRH deficiency were also detected in several patients. In each of 3 different patients with an X-linked mode of inheritance, 3 genetic defects, 2 point mutations and a small intragenic deletion, were detected. These defects consist of a single base mutation introducing a stop codon at position 328, a single base mutation resulting in a phenylalanine to leucine substitution at position 517, and a 9-base deletion at the 3'-exon-intron splice site of exon 8, respectively. All identified genetic defects occur within the fibronectin type III repeats of the predicted protein encoded by the KAL-1 gene. In conclusion, our study indicates that the incidence of genetic defects within the coding region of the KAL-1 gene in patients with sporadic GnRH deficiency is low (5-8%), thus supporting the idea that the X-linked form of inheritance represents the least common form of the disease.

Genetic Heterogeneity Evidenced by Low Incidence of KAL-1 Gene Mutations in Sporadic Cases of Gonadotropin-Releasing Hormone Deficiency

Genetic Heterogeneity Evidenced by Low Incidence of KAL-1 Gene Mutations in Sporadic Cases of Gonadotropin-Releasing Hormone Deficiency

Initial characterization of anosmin-1, a putative extracellular matrix protein synthesized by definite neuronal cell populations in the central nervous system.

The KAL gene is responsible for the X-chromosome linked form of Kallmann's syndrome in humans. Upon transfection of CHO cells with a human KAL cDNA, the corresponding encoded protein, KALc, was produced. This protein is N-glycosylated, secreted in the cell culture medium, and is localized at the cell surface. Several lines of evidence indicate that heparan-sulfate chains of proteoglycan(s) are involved in the binding of KALc to the cell membrane. Polyclonal and monoclonal antibodies to the purified KALc were generated. They allowed us to detect and characterize the protein encoded by the KAL gene in the chicken central nervous system at late stages of embryonic development. This protein is synthesized by definite neuronal cell populations including Purkinje cells in the cerebellum, mitral cells in the olfactory bulbs and several subpopulations in the optic tectum and the striatum. The protein, with an approximate molecular mass of 100 kDa, was named anosmin-1 in reference to the deficiency of the sense of smell which characterizes the human disease. Anosmin-1 is likely to be an extracellular matrix component. Since heparin treatment of cell membrane fractions from cerebellum and tectum resulted in the release of the protein, we suggest that one or several heparan-sulfate proteoglycans are involved in the binding of anosmin-1 to the membranes in vivo.

Characterization of the promoter of the human KAL gene, responsible for the X-chromosome-linked Kallmann syndrome

We report on the first characterization of the human KAL promoter ( pKAL), based on the analysis of a 2-kb fragment of the 5′ flanking region. As determined by primer extension, transcription of the human KAL gene is initiated at two different sites in the quail embryonic neuroretina QNR/D cell line. The promoter region is G + C rich and contains a CCAAT box, two binding sites for the SP1 transcription factor and two AP2-binding sites, but no TATA box. It also shares a motif with several neural-specific genes. The ability of four deletion mutants to drive transcription of the heterologous chloramphenicol acetyltransferase (CAT)-encoding gene was determined in transfection experiments. The mutant containing the KAL sequence from nt + 2 to −435 demonstrated a tissue-specific, although weak, transcriptional activity only in the quail embryonic neuroretina K2 and QNR/D cell lines. Longer constructs did not confer any activity. Therefore, we suggest that this 437-bp segment of pKAL constitutes a neural-specific promoter which could be negatively controlled by upstream sequences.

Variable penetrance of hypogonadism in a sibship with Kallmann syndrome due to a deletion of the KAL gene

We report on the clinical and molecular characterization of 3 sibs with X-linked ichthyosis and variable expression of Kallmann syndrome. One of the affected brothers had mild hyposmia and showed normal pubertal progression. However, we demonstrated the same partial deletion of the X-linked Kallmann gene, sparing the first exon in the mildly affected patient as well as in one of his severely affected brothers.

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.

Diagnosis of Kallmann's Syndrome at Eighteen Months of Age by Demonstration of Base Substitution in Kal Gene by Direct PCR-Sequencing

Diagnosis of Kallmann's Syndrome at Eighteen Months of Age by Demonstration of Base Substitution in Kal Gene by Direct PCR-Sequencing

KAL, a gene mutated in Kallmann's syndrome, is expressed in the first trimester of human development

Kallmann's syndrome (KS) is characterised by the association of anosmia and isolated hypogonadotrophic hypogonadism (IHH). Mutations of the KAL gene which is located at Xp22.3 cause X-linked KS (XKS). In this study we used the reverse transcriptase polymerase chain reaction and in situ hybridisation to examine the developmental expression of KAL in the first trimester of pregnancy, the earliest stage of human gestation examined thus far. At 45 days after fertilisation KAL mRNA was detected in the spinal cord, the mesonephros and metanephros but not in the brain. Later in gestation, at 11 weeks, the gene was expressed in the developing olfactory bulb, retina and kidney. This expression pattern correlates with the clinical findings in XKS since olfactory bulb dysgenesis with subsequent defective neural migration causes anosmia and IHH. Additionally, renal agenesis occurs in 40% of patients. Therefore this study provides strong evidence that KAL expression is required for the normal development of the olfactory bulb and kidney in the first trimester of human pregnancy.

Analysis of the kal gene in males and females with idiopathic hypogonadotropic hypogonadism

Analysis of the kal gene in males and females with idiopathic hypogonadotropic hypogonadism

Early expression of the KAL gene during embryonic development of the chick.

The human KAL gene is responsible for the X chromosome-linked Kallmann syndrome, which consists of the association of hypogonadotropic hypogonadism and anosmia. The human and chicken KAL genes have been isolated. Using in situ hybridization, we studied KAL gene expression during development of the chick. We have previously reported that, from embryonic day 8, the expression is almost restricted to definite neuronal populations in the central nervous system, most of which still express the gene after hatching. Here we report that the KAL gene is also expressed during early embryonic development (days 2-8) in various endodermal, mesodermal, and neurectodermal derivatives. In most endodermal and mesodermal derivatives, the expression is transient and precedes cell differentiation. In contrast, the expression in the nervous system concerns postmitotic central neuroblastic populations, most of which still express the gene after differentiation. In accordance with such a spatio-temporal pattern of expression, we suggest that the KAL gene is involved both in morphogenetic events and in neuronal late differentiation. In addition, the absence of detectable expression of the KAL gene either in the embryonic olfactory epithelium or in the surrounding nasal mesenchyme reinforces the hypothesis that Kallmann's syndrome results from a central olfactory target cell defect.

Isolation and characterization of the gene responsible for the X chromosome-linked Kallmann syndrome

Kallmann de Morsier Syndrome is defined by the association of an hypogonadism with an anosmia. The hypogonadism is due to a deficiency of GnRH (gonadotropin-releasing hormone). Olfactory bulbs and tracts are under-developed in the patients. Embryological studies have indicated that the migration of GnRH neurons and the axonal extension of olfactory neurons, which both originate in the olfactory epithelium during embryogenesis, were impaired in a fetus affected by X-linked Kallmann Syndrome. By a positional cloning strategy, we have isolated the KAL gene, responsible for the X-linked form of the disease. The gene consists of 14 exons. A highly homologous pseudogene on the Y chromosome has been characterized. The KAL gene encodes a putative secreted protein of 680 amino acids, which contains four fibronectin type III repeats and a four disulphide core motif. The former motif is usually associated with adhesion function. The latter has been described in protein with antiprotease activity. We have isolated the chicken KAL homologue and studied its expression by in situ hybridization during late embryonic development. The gene is expressed in various neuronal populations of the central nervous system, including mitral cells of the olfactory bulbs. We suggested that the KAL protein might be involved in late neuronal differentiation.

Expression of the Kallmann syndrome gene in human fetal brain and in the manipulated chick embryo.

Kallmann syndrome is an inherited disorder characterized by an abnormality in olfactory system development. The gene for the X-linked form of this disorder (KAL) maps to Xp22.3 and encodes a protein sharing homologies with molecules involved in neuronal migration and axonal pathfinding. Here we report the expression pattern of the KAL gene in various parts of the human fetal brain. We found KAL transcripts in granule cells of the olfactory bulb and the cerebellum, in the dorsomedial thalamus and in the developing cerebral cortex. To determine whether or not signals from the olfactory nerve are required for KAL expression in the olfactory bulb, we analyzed chick embryos in which the olfactory placode was surgically removed. Those embryos lacking an olfactory nerve had a histologically abnormal bulb which nevertheless expressed the KAL gene at high levels. These findings indicate that, while the development of the proper cytoarchitecture of the olfactory bulb requires the innervation by olfactory axons, the expression of KAL is independent of such developmental processes.