A frameshift mutation in prominin (mouse)-like 1 causes human retinal degeneration.

The disks of vertebrate photoreceptors are produced by outgrowths of the plasma membrane. Hence genes that encode retinal proteins targeted to plasma membrane protrusions represent candidates for inherited retinal degenerations. One such candidate is the gene encoding human prominin (mouse)-like 1 (PROML1, previously known as AC133 antigen) which belongs to the prominin family of 5-transmembrane domain proteins. Murine prominin (prom) shows a strong preference for plasma membrane protrusions in a variety of epithelial cells whereas PROML1 is expressed in retinoblastoma cell lines and adult retina. In the present study, molecular genetic analyses of a pedigree segregating for autosomal recessive retinal degeneration indicated that the affected individuals were homozygous for a nucleotide 1878 deletion in PROML1. This alteration is predicted to result in a frameshift at codon 614 with premature termination of translation. Expression of a similar prom deletion mutant in CHO cells indicated that the truncated protein does not reach the cell surface. Immunocytochemistry revealed that prom is concentrated in the plasma membrane evaginations at the base of the outer segments of rod photoreceptors. These findings suggest that loss of prominin causes retinal degeneration, possibly because of impaired generation of the evaginations and/or impaired conversion of the evaginations to disks.

Connexin26 deafness in several interconnected families.

Mutations in the connexin26 gene are the basis of much autosomal recessive sensorineural deafness. There is a high frequency of mutant alleles, largely accounted for by one common mutation, 35delG. We have studied a group of families, who had been brought together through marriages between Deaf persons, in which there are more than 30 Deaf people in four generations. We show that many of the several cases of deafness are the result of 35delG homozygosity or 35delG/Q57X compound heterozygosity at the connexin26 locus. A considerable range of audiographic phenotypes was observed. The combined effects of a high population frequency of mutant alleles, and of positive assortative marriage among the Deaf, led to an infrequently observed recessive pedigree pattern.

The deafness-associated mitochondrial DNA mutation at position 7445, which affects tRNASer(UCN) precursor processing, has long-range effects on NADH dehydrogenase subunit ND6 gene expression.

The pathogenetic mechanism of the deafness-associated mitochondrial DNA (mtDNA) T7445C mutation has been investigated in several lymphoblastoid cell lines from members of a New Zealand pedigree exhibiting the mutation in homoplasmic form and from control individuals. We show here that the mutation flanks the 3' end of the tRNASer(UCN) gene sequence and affects the rate but not the sites of processing of the tRNA precursor. This causes an average reduction of approximately 70% in the tRNASer(UCN) level and a decrease of approximately 45% in protein synthesis rate in the cell lines analyzed. The data show a sharp threshold in the capacity of tRNASer(UCN) to support the wild-type protein synthesis rate, which corresponds to approximately 40% of the control level of this tRNA. Strikingly, a 7445 mutation-associated marked reduction has been observed in the level of the mRNA for the NADH dehydrogenase (complex I) ND6 subunit gene, which is located approximately 7 kbp upstream and is cotranscribed with the tRNASer(UCN) gene, with strong evidence pointing to a mechanistic link with the tRNA precursor processing defect. Such reduction significantly affects the rate of synthesis of the ND6 subunit and plays a determinant role in the deafness-associated respiratory phenotype of the mutant cell lines. In particular, it accounts for their specific, very significant decrease in glutamate- or malate-dependent O2 consumption. Furthermore, several homoplasmic mtDNA mutations affecting subunits of NADH dehydrogenase may play a synergistic role in the establishment of the respiratory phenotype of the mutant cells.

Mitochondrial A7445G mutation in two pedigrees with palmoplantar keratoderma and deafness.

A New Zealand and a Scottish pedigree with maternally inherited sensorineural deafness were both previously shown to carry a heteroplasmic A7445G mutation in the mitochondrial genome. More detailed clinical examination of the New Zealand family showed that the hearing loss was progressive, with the severity of the overall loss and the frequencies most affected differing markedly between individuals of similar age, and showed that many relatives also had palmoplantar keratoderma. Review of the literature demonstrated three other large families with presumed autosomal dominant inheritance of palmoplantar keratoderma and hearing loss. In a United Kingdom pedigree the syndrome was transmitted by female and male parents, an inheritance pattern which made mitochondrial inheritance unlikely; however, in a Turkish and a Japanese pedigree the affected individuals were all maternally related. Subsequent analysis of the Japanese pedigree documented the same A7445G mitochondrial mutation as was previously found in the New Zealand and Scottish pedigrees. Other mitochondrial sequence variants previously reported in the New Zealand or Scottish pedigrees were absent from the Japanese pedigree which suggests that the A7445G mutation arose independently in all three pedigrees. To our knowledge palmoplantar keratoderma has not previously been associated with mitochondrial defects; however, the current findings suggest that the A7445G mutation is associated not only with progressive hearing loss but also with palmoplantar keratoderma. The penetrance and expressivity of both symptoms varied considerably between individuals in the Scottish and New Zealand Studies which suggests that additional environmental and/or genetic factors are involved.

Mutation of the gene encoding cellular retinaldehyde-binding protein in autosomal recessive retinitis pigmentosa.

Inadequate levels of all-trans-retinol in the blood cause retinal dysfunction; hence, genes implicated in retinal vitamin-A metabolism represent candidates for inherited retinal degenerations. In the current study, molecular genetic analysis of a consanguineous pedigree segregating for non-syndromic autosomal recessive retinitis pigmentosa (arRP) indicated that the affected siblings were homozygous by descent for a G4763A nucleotide substitution in RLBP1, the gene encoding cellular retinaldehyde-binding protein (CRALBP). This substitution is predicted to replace an arginine with glutamine at residue 150. CRALBP is not expressed in photoreceptors but is abundant in the retinal pigment epithelium (RPE) and Müller cells of the neuroretina, where it carries 11-cis-retinol and 11-cis-retinaldehyde. When expressed in bacteria, recombinant CRALBP (rCRALBP) containing the R150Q substitution was less soluble than wild-type rCRALBP. Mutant rCRALBP was purified from the soluble cell lysate and the protein structure was verified by mass spectrometry. The mutant protein lacked the ability to bind 11-cis-retinaldehyde. These findings suggest that arRP in the current pedigree results from a lack of functional CRALBP, presumably leading to disruption of retinal vitamin-A metabolism.

Prelingual deafness: high prevalence of a 30delG mutation in the connexin 26 gene.

Prelingual non-syndromic (isolated) deafness is the most frequent hereditary sensory defect. In >80% of the cases, the mode of transmission is autosomal recessive. To date, 14 loci have been identified for the recessive forms (DFNB loci). For two of them, DFNB1 and DFNB2, the genes responsible have been characterized; they encode connexin 26 and myosin VIIA, respectively. In order to evaluate the extent to which the connexin 26 gene (Cx26) contributes to prelingual deafness, we searched for mutations in this gene in 65 affected Caucasian families originating from various countries, mainly tunisia, France, New Zealand and the UK. Six of these families are consanguineous, and deafness was shown to be linked to the DFNB1 locus, 10 are small non consanguineous families in which the segregation of the trait has been found to be compatible with the involvement of DFNB1, and in the remaining 49 families no linkage analysis has been performed. A total of 62 mutant alleles in 39 families were identified. Therefore, mutations in Cx26 represent a major cause of recessively inherited prelingual deafness since according to the present results they would underlie approximately half of the cases. In addition, one specific mutation, 30delG, accounts for the majority (approximately 70%) of the Cx26 mutant alleles. It is therefore one of the most frequent disease mutations so far identified. Several lines of evidence indicate that the high prevalence of the 30delG mutation arises from a mutation hot spot rather than from a founder effect. Genetic counseling for prelingual deafness has been so far considerably impaired by the difficulty in distinguishing genetic and non genetic deafness in families presenting with a single deaf child. Based on the results presented here, the development of a simple molecular test could be designed which should be of considerable help.

The contribution of the DFNB1 locus to neurosensory deafness in a Caucasian population.

Classical studies have demonstrated genetic heterogeneity for nonsyndromic autosomal recessive congenital neurosensory deafness, with at least six loci postulated. Linkage analysis in two consanguineous Tunisian kindreds has demonstrated that one such deafness locus, DFNB1, maps near chromosome 13 markers D13S175, D13S143, and D13S115. We tested these markers for cosegregation with deafness in 18 New Zealand and 1 Australian nonconsanguineous kindreds, each of which included at least two siblings with nonsyndromic presumed congenital sensorineural deafness and that had a pedigree structure consistent with autosomal recessive inheritance. When all families were combined, a peak two-point lod score of 2.547 (theta = .1) was obtained for D13S175, 0.780 (theta = .2) for D13S143, and 0.664 (theta = .3) for D13S115. While there was no statistically significant evidence for heterogeneity at any of the three loci tested, nine families showed cosegregation of marker haplotypes with deafness. These observations suggest that the DFNB1 locus may make an important contribution to autosomal recessive neurosensory deafness in a Caucasian population. In the nine cosegregating families, phenotypic variation was observed both within sibships (in four families), which indicates that variable expressivity characterizes some genotypes at the DFNB1 locus, and between generations (in two families), which suggests allelic heterogeneity.

Oguchi disease: suggestion of linkage to markers on chromosome 2q.

Oguchi disease is a rare autosomal recessive form of congenital stationary night blindness. The condition is associated with fundus discolouration and abnormally slow dark adaptation. Earlier studies suggested that the 48 kD protein S antigen may be involved in the recovery phase of light transduction. Previous cytogenetic and linkage studies have localised the S antigen gene (SAG) to chromosome 2q37.1. In the present study markers which map to distal chromosome 2q were typed in an inbred Oguchi pedigree. The segregation data obtained suggested that the affected subjects are homozygous by descent for a region between D2S172 and D2S345. An intragenic SAG polymorphism was homozygous in all affected people and a recombination event suggested that SAG maps proximal to D2S345. Collectively, these findings support the hypothesis that a defect in S antigen may be responsible for Oguchi disease.

Relaxation of insulin-like growth factor II gene imprinting implicated in Wilms' tumour.

Genomic imprinting has been implicated in the onset of several embryonal tumours but the mechanism is not well understood. Maternal chromosome 11p15 loss of heterozygosity and paternal chromosome 11 isodisomy suggest that imprinted genes are involved in the onset of Wilms' tumour and the Beckwith-Wiedemann syndrome. The insulin-like growth factor II (IGF2) gene located at 11p15.5 has been put forward as a candidate gene as it is maternally imprinted (paternally expressed) in the mouse, and is expressed at high levels in Wilms' tumours. We report here that the IGF2 gene is expressed from the paternal allele in human fetal tissue, but that in Wilms' tumour expression can occur biallelically. These results provide, to our knowledge, the first evidence that relaxation of imprinting may play a role in the onset of disease and suggest a new genetic mechanism involved in the development of cancer.

A third Wilms' tumor locus on chromosome 16q.

Loss of heterozygosity studies have been used to identify chromosomal regions which are frequently deleted and thus indicate areas which may harbor tumor suppressor genes. As a result, both the WT1 gene located in chromosome 11p13 and an unidentified gene(s) within chromosome 11p15 have been implicated in Wilms' tumorigenesis. Cytogenetic and linkage studies suggest that additional non-chromosome 11 sites are involved in Wilms' tumor. Because these sites may also involve loss of heterozygosity, loci on 33 autosomal arms were screened for allele loss in a series of Wilms' tumors. We found that in addition to loss on chromosome 11p (11 of 25 informative tumors) there was significant loss on chromosome 16q (9 of 45 informative tumors), while the total frequency of allele loss excluding these loci was low (9 of 426 total informative loci). These data indicate that losses of both chromosome 11p and 16q alleles are nonrandom events and suggest that 16q is the location of a third tumor suppressor gene underlying Wilms' tumorigenesis. The parental origin of the lost chromosome 16q allele was determined in eight sporadic tumors. Alleles of paternal and of maternal origin were each lost in four sporadic tumors indicating that, unlike chromosome 11p, alleles of either parental origin are lost on 16q.