A non-syndrome form of neurosensory, recessive deafness maps to the pericentromeric region of chromosome 13q.

Non-syndromic, recessively inherited deafness is the most predominant form of severe inherited childhood deafness. Until now, no gene responsible for this type of deafness has been localized, due to extreme genetic heterogeneity and limited clinical differentiation. Linkage analyses using highly polymorphic microsatellite markers were performed on two consanguineous families from Tunisia affected by this form of deafness. The deafness was profound, fully penetrant and prelingual. A maximum two-point lod score of 9.88 (theta = 0.001) was found with a marker detecting a 13q locus (D13S175). Linkage was also observed to the pericentromeric 13q12 loci D13S115 and D13S143. These data map this neurosensory deafness gene to the same region of chromosome 13q as the gene for severe, childhood autosomal recessive muscular dystrophy.

Xp22.3 deletions in isolated familial Kallmann's syndrome.

Several familial cases of Kallmann's syndrome (KS) have been reported, among which the X-chromosome-linked mode of inheritance is the most frequent. The gene responsible for the X-linked KS has been localized to the terminal part of the X-chromosome short arm (Xp22.3 region), immediately proximal to the steroid sulfatase gene responsible for X-linked ichthyosis. Large deletions of this region have been previously shown in patients affected with both X-linked ichthyosis and KS. We report here the search for Xp22.3 deletions in 20 unrelated males affected with isolated X-linked KS. Only 2 deletions were found using Southern blot analysis, indicating that large deletions are uncommon in patients affected with KS alone. Both deletions were shown to include the entire KAL gene responsible for X-linked KS. The patients carrying these deletions exhibit additional clinical anomalies, which are discussed: unilateral renal aplasia, unilateral absence of vas deferens, mirror movements, and sensory neural hearing loss.

No evidence for linkage to the type 1 or type 2 neurofibromatosis loci in Noonan syndrome families.

A linkage analysis has been performed on 6 two-generation families with classical Noonan syndrome to determine whether the syndrome is linked to neurofibromatosis type 1 on chromosome 17q or to neurofibromatosis type 2 on chromosome 22q. A significantly negative location score was obtained between 10 cM centromeric to and 15 cM telomeric from the neurofibromatosis type 1 locus. A significantly negative lod score was obtained with a marker mapping within the region where neurofibromatosis type 2 is thought to be located. These data indicate that Noonan syndrome is not tightly linked to either neurofibromatosis type 1 or type 2.

Differentiation is not restored in hybrids between independent variants of a rat hepatoma.

Crosses have been undertaken between cells of three independent clones of dedifferentiated rat hepatoma variants to investigate whether "complementation" leading to restoration of the original differentiation would occur. Hybrids were examined between ten days and two months after fusion for the presence of intracellular albumin and for their ability to proliferate in glucose-free medium where survival requires activity of the liver-specific gluconeogenic enzymes. In none of the three possible crosses involving the three variants was evidence of reexpression of hepatic functions obtained.

Physical mapping of the human pseudo-autosomal region; comparison with genetic linkage map.

A long-range restriction map of the pseudo-autosomal or exchange pairing region (corresponding to the terminal parts of the short arms of the human sex chromosomes) has been established using pulsed field gel electrophoresis. A total of seven loci have been located on this physical map based essentially on the analysis of 45,X Turner genomes. The region spans a total of 2600 kb. The 5' end of the MIC2 gene maps at less than 80 kb from the proximal pseudo-autosomal boundary. Since the total pseudo-autosomal linkage interval represents approximately 50% of recombination at male meiosis, 1 cM corresponds to 50-60 kb. This is consistent with the almost 20-fold increase in recombination frequency observed in male versus female meiosis in this region. The present data show no distortion between both physical and linkage maps. The distribution of the CpG-rich restriction sites is notably disequilibrated. A large subset of these sites is concentrated within the 500 kb closest to the telomere whereas others appear in clusters (probably HTF islands) scattered in the rest of the pseudo-autosomal region.

Long-range restriction map of the terminal part of the short arm of the human X chromosome.

The terminal part of the short arm of the human X chromosome has been mapped by pulsed-field gel electrophoresis (PFGE). The map, representing the distal two-thirds of Xp22.3 spans a total of 10,000 kilobases (kb) from Xpter to the DXS143 locus. A comparison with linkage data indicates that 1 centimorgan (cM) in this region corresponds to about 600 kb. CpG islands were essentially concentrated in the 1500 kb immediately proximal to the pseudoautosomal boundary. Several loci, including the gene encoding steroid sulfatase (STS) and the loci for the X-linked recessive form of chondrodysplasia punctata (CDPX) and for Kallmann syndrome (KAL) have been placed relative to the Xp telomere. CDPX is located between 2650 and 5550 kb from Xpter, and STS is located between 7250 and 7830 kb from Xpter. KAL maps to an interval of 350 kb between 8600 and 8950 kb from the telomere. The X-chromosomal breakpoints of a high proportion of XX males resulting from X-Y interchange cluster to a 920-kb region proximal and close to the pseudoautosomal boundary.

Exchange of terminal portions of X- and Y-chromosomal short arms in human XY females.

Human Y(+) XX maleness has been shown to result from an abnormal terminal Xp-Yp interchange that can occur during paternal meiosis. To test whether human XY females are produced by the same mechanism, we followed the inheritance of paternal pseudoautosomal loci and Xp22.3-specific loci in two XY female patients. Y-specific sequences and the whole pseudoautosomal region of the Y chromosome of their fathers were absent in these patients. However, the entire pseudoautosomal region and the X-specific part of Xp22.3 distal to the STS locus had been inherited from the X chromosome of the respective father. This Xp transfer to Yp was established by in situ hybridization experiments showing an Xp22.3-specific locus on Yp in both cases. Such results demonstrate that an abnormal and terminal X-Y interchange generated the rearranged Y chromosome of these two XY females; they appear to be the true countertype of Y(+) XX males. In these patients, who also display some Turner stigmata, the Y gene(s) involved in this phenotype is (are) localized to interval 1 or 2. If the loss of such gene(s) affects fetal viability, their proximity to TDF would account for the underrepresentation of interchange 46,XY females compared with Y(+) XX males.

Molecular genetics of hearing loss.

Hereditary isolated hearing loss is genetically highly heterogeneous. Over 100 genes are predicted to cause this disorder in humans. Sixty loci have been reported and 24 genes underlying 28 deafness forms have been identified. The present epistemic stage in the realm consists in a preliminary characterization of the encoded proteins and the associated defective biological processes. Since for several of the deafness forms we still only have fuzzy notions of their pathogenesis, we here adopt a presentation of the various deafness forms based on the site of the primary defect: hair cell defects, nonsensory cell defects, and tectorial membrane anomalies. The various deafness forms so far studied appear as monogenic disorders. They are all rare with the exception of one, caused by mutations in the gene encoding the gap junction protein connexin26, which accounts for between one third to one half of the cases of prelingual inherited deafness in Caucasian populations.

Tissue-specific expression of the rat albumin gene: genetic control of its extinction in microcell hybrids.

Numerous studies of cell hybrids have indicated that somatic cells produce negative regulators (extinguishers) that prevent the expression of functions foreign to their own differentiation. Here, we report genetic evidence of such control. In microcell hybrids between well-differentiated rat hepatoma cells and microcells of mouse fibroblast L cells, the extinction of albumin synthesis is directly related to the presence of a single specific chromosome of the mouse fibroblast parent. The expression of several other hepatic functions is not affected. Transfection of these hybrids with a recombinant plasmid, containing a tissue-specific control element of the upstream region of the rat albumin gene linked to coding sequences of the chloramphenicol acetyltransferase gene, reveals that extinction acts on or via this cis-control element.

Isolation of sequences from Xp22.3 and deletion mapping using sex chromosome rearrangements from human X-Y interchange sex reversals.

A repeated DNA element (STIR) interspersed in Xp22.3 and on the Y chromosome has been used as a tag to isolate seven single-copy probes from the human sex chromosomes. The seven probes detect X-specific loci located in Xp22.3. Using a panel of X-chromosomal deletions from X-Y interchange sex reversals (XX males and XY females), these X-specific loci and some additional ones were mapped to four contiguous intervals of Xp22.3, proximal to the pseudoautosomal region and distal to STS. The construction of this deletion map of the terminal part of the human X chromosome can serve as a starting point for a long-range physical map of Xp22.3 and for a more accurate mapping of genetic diseases located in Xp22.3.

An interstitial deletion in Xp22.3 in a family with X-linked recessive chondrodysplasia punctata and short stature.

In a four-generation family, chondrodysplasia punctata was found in a boy and one of his maternal uncles. These two patients also have short stature, as do all female members of the family, DNA molecular analysis of the pseudoautosomal and Xp22.3-specific loci revealed the presence of an interstitial deletion that cosegregates with the phenotypic abnormalities. The proximal breakpoint of this deletion was located distal to the DXS31 locus and the distal breakpoint in the pseudoautosomal region between DXYS59 and DXYS17. This maps the recessive X-linked form of chondrodysplasia punctata between the proximal boundary of the pseudoautosomal region and DXS31, and an Xp gene controlling growth between DXYS59 and DXS31.

An abnormal terminal X-Y interchange accounts for most but not all cases of human XX maleness.

To determine if human XX maleness results from an abnormal chromosomal X-Y interchange, we studied the inheritance of the paternal pseudoautosomal region in nine patients. Those six patients in whom Y-specific DNA was found (Y(+)) inherited the entire pseudoautosomal region from the paternal Y chromosome and lost that of the paternal X chromosome. Moreover, in three Y(+) cases, we observed the deletion of a paternal Xp locus tightly linked to the pseudoautosomal region. These results definitively show that an abnormal and terminal X-Y interchange during paternal meiosis causes Y(+)XX maleness. In contrast, no abnormal X-Y interchange was observed in any of the three Y(-) cases analyzed, suggesting that maleness can occur in the absence of any Y-specific DNA.

Normal and abnormal interchanges between the human X and Y chromosomes.

A single obligatory recombination event takes place at male meiosis in the tips of the X- and Y-chromosome short arms (i.e. the pseudoautosomal region). The crossover point is at variable locations and thus allows recombination mapping of the pseudoautosomal loci along a gradient of sex linkage. Recombination at male meiosis in the terminal regions of the short arms of the X and Y chromosomes is 10- to 20-fold higher than between the same regions of the X chromosomes during female meiosis. The human pseudoautosomal region is rich in highly polymorphic loci associated with minisatellites. However, these minisatellites are unrelated to those resembling the bacterial Chi sequence and which possibly represent recombination hotspots. The high recombination activity of the pseudoautosomal region at male meiosis sometimes results in unequal crossover which can generate various sex-reversal syndromes.

Mutational analysis of SRY: nonsense and missense mutations in XY sex reversal.

XY females (n = 17) were analysed for mutations in SRY (sex-determining region Y gene), a gene that has recently been equated with the testis determining factor (TDF). SRY sequences were amplified by the polymerase chain reaction (PCR) and analysed by both the single strand conformational polymorphism assay (SSCP) and DNA sequencing. The DNA from two individuals gave altered SSCP patterns; only these two individuals showed any DNA sequence variation. In both cases, a single base change was found, one altering a tryptophan codon to a stop codon, the other causing a glycine to arginine amino acid substitution. These substitutions lie in the high mobility group (HMG)-related box of the SRY protein, a potential DNA-binding domain. The corresponding regions of DNA from the father of one individual and the paternal uncle of the other, were sequenced and found to be normal. Thus, in both cases, sex reversal is associated with de novo mutations in SRY. Combining this data with two previously published reports, a total of 40 XY females have now been analysed for mutations in SRY. The number of de novo mutations in SRY is now doubled to four, adding further strength to the argument that SRY is TDF.

Prevalence and molecular analysis of two hot spots for ectopic recombination leading to XX maleness.

Two hot spots of ectopic Xp-Yp recombination have previously been shown to be at the origin of XX maleness (Weil et al., 1994, Nature Genet. 7:414-419). To get more insight into the molecular basis of the abnormal interchange, 25 Y(+) class 3 XX male patients were studied. The two hot spots were found to account for the aberrant exchange in more than 50% of the cases. In addition, there is a correlation between the prevalence of each hot spot and the degree of X-Y homology between the corresponding fragments. Sequencing of the X-Y junctions in six patients, who carried a breakpoint mapping in either of these two hot spot fragments, showed that their precise locations were different from one individual to the other. In particular, the results obtained here in four new patients exclude the possibility that the repeated elements, present in these X-Y homologous fragments, are responsible for the high incidence of X-Y interchanges observed. Moreover, the breakpoints of all 25 class 3 XX males were found to be arranged in the same order on the X and Y chromosomes. This suggests that most ectopic recombinations leading to class 3 XX maleness involve X-Y homologous sequences persisting from an ancestral larger block of homology on the short arms of both sex chromosomes.

Sequence characterization of a newly identified human alpha-tubulin gene (TUBA2).

We report on the isolation and initial characterization of a human alpha-tubulin gene named TUBA2. This gene is located in the 13q11 region and has been considered a candidate gene for two nonsyndromic deafnesses, DFNB1 and DFNA3. The gene, with a minimum size of 6.5 kb, contains five exons and four introns starting at codon positions 1, 76, 125, and 352, one of which is inserted between the initiation methionine codon and the codon specifying the second amino acid, arginine 2. Neither rearrangement nor point mutation was found in the coding region of the gene in DFNB1- and DFNA3-affected patients. The gene was therefore unlikely to be responsible for either of these deafnesses. During the characterization of TUBA2, the gene encoding connexin 26 was proven to be responsible for both DFNB1 and DFNA3 (D. P. Kelsell et al., 1997, Nature 387: 80-83). However, the present data offer the possibility of testing the involvement of the TUBA2 gene in the Clouston hidrotic ectodermal dysplasia and the Kabuki syndrome, two genetic diseases that have recently been mapped to the 13q11 region.

Anosmin-1 underlying the X chromosome-linked Kallmann syndrome is an adhesion molecule that can modulate neurite growth in a cell-type specific manner.

Anosmin-1 is an extracellular matrix glycoprotein which underlies the X chromosome-linked form of Kallmann syndrome. This disease is characterized by hypogonadism due to GnRH deficiency, and a defective sense of smell related to the underdevelopment of the olfactory bulbs. This study reports that anosmin-1 is an adhesion molecule for a variety of neuronal and non-neuronal cell types in vitro. We show that cell adhesion to anosmin-1 is dependent on the presence of heparan sulfate and chondroitin sulfate glycosaminoglycans at the cell surface. A major cell adhesion site of anosmin-1 was identified in a 32 amino acid (32R1) sequence located within the first fibronectin-like type III repeat of the protein. The role of anosmin-1 as a substrate for neurite growth was tested on either coated culture dishes or monolayers of anosmin-1-producing CHO cells. In both experimental systems, anosmin-1 was shown to be a permissive substrate for the neurite growth of different types of neurons. Mouse P5 cerebellar neurons cultured on anosmin-1 coated wells developed long neurites; the 32R1 peptide was found to underly part of this neurite growth activity. When the cerebellar neurons were cultured on anosmin-1-producing CHO cells, neurite growth was reduced as compared to wild-type CHO cells; in contrast, no difference was observed for E18 hippocampal and P1 dorsal root ganglion neurons in the same experimental system. These results indicate that anosmin-1 can modulate neurite growth in a cell-type specific manner. Finally, anosmin-1 induced neurite fasciculation of P5 cerebellar neuron aggregates cultured on anosmin-1-producing CHO cells. The pathogenesis of the olfactory defect in the X-linked Kallmann syndrome is discussed in the light of the present results and the recent data reporting the immunohistochemical localisation of anosmin-1 during early embryonic development.