A gene for congenital, recessive deafness DFNB3 maps to the pericentromeric region of chromosome 17.

Two percent of the residents of Bengkala, Bali, have profound, congenital, neurosensory, nonsyndromal deafness due to an autosomal recessive mutation at the DFNB3 locus. We have employed a direct genome-wide disequilibrium search strategy, allele-frequency-dependent homozygosity mapping (AHM), and an analysis of historical recombinants to map DFNB3 and position the locus relative to flanking markers. DFNB3 maps to chromosome 17, closest to D17S261, pRM7-GT and D17S805. In individuals homozygous for DFNB3, historical recombinant genotypes for the flanking markers, D17S122 and D17S783, place DFNB3 in a 5.3 cM interval of the pericentromeric region of chromosome 17 on a refined linkage map of 17p-17q12. Based on conserved synteny, the murine sh2 gene may be the homologue of DFNB3.

Human inner ear OCP2 cDNA maps to 5q22-5q35.2 with related sequences on chromosomes 4p16.2-4p14, 5p13-5q22, 7pter-q22, 10 and 12p13-12qter.

Mouse Ocp2-rs2 maps to chromosome 11 and encodes an 18.6 kDa peptide abundantly expressed in the organ of Corti. We show that sequences similar to murine Ocp2-rs2 are found on human chromosomes 4p16.2-4p14, 5p13-5q35.2, 7pter-q22, 10 and 12p13-12qter as revealed by Southern blot analyses of human/rodent somatic cell hybrids. A fetal human inner ear cDNA library was screened with a cloned 254 bp PCR product of murine Ocp2-rs2. One of two human cDNA clones (CM1) was sequenced from the 5' end that begins with murine Ocp2-rs2 codon 14 through the stop codon and 258 nucleotides of 3-UTR and was found to have the identical deduced amino acid sequence to Ocp2-rs2. Based on the sequence in the 3'-UTR of CM1, a PCR primer pain was synthesized and used to confirm that a human homologue of Ocp2-rs2, designated OCP2 and expressed in the developing human inner ear, is localized to 5q22-5q35.2. Other OCP2-like sequences located on chromosomes 4p16.2-4p14, 7pter-q22 and 12p13-12qter (but not the chromosome 10 OCP2-like sequence) will PCR amplify the expected size product at a lower annealing temperature using the OCP2 3'-UTR PCR primers indicating that there may be a human OCP2 gene family.

Autosomal dominant microcephaly with normal intelligence, short palpebral fissures, and digital anomalies.

We describe a family segregating an autosomal dominant mutation producing a syndrome comprising microcephaly with normal intelligence and short palpebral fissures together with variable signs including thumb hypoplasia, shortness of the middle phalanges of the second and fifth fingers, small feet, a gap between the first and second toes, and mild syndactyly of the toes or fingers. A characteristic radiologic finding in our family is thinning of the proximal end of the first metacarpal and shortening of that metacarpal. The severity of these findings was asymmetric in our patients. This syndrome is similar to patients described by Brunner and Winter [1991: J Med Genet 28: 389-394], Feingold [1975: Synd Ident 3:16-17, 1978: Hosp Prac 13:44-49], and König et al. [1990: Dysmorphol Clin Genet 4:83-86].

Exclusion of BMP6 as a candidate gene for cleidocranial dysplasia.

Cleidocranial dysplasia (CCD) is an autosomal dominant, generalized skeletal dysplasia in humans that has been mapped to the short arm of chromosome 6. We report linkage of a CCD mutation to 6p21 in a large family and exclude the bone morphogenetic protein 6 gene (BMP6) as a candidate for the disease by cytogenetic localization and genetic recombination. CCD was linked with a maximal two-point LOD score of 7.22 with marker D6S452 at theta = 0. One relative with a recombination between D6S451 and D6S459 and another individual with a recombination between D6S465 and CCD places the mutation within a 7 cM region between D6S451 and D6S465 at 6p21. A phage P1 genomic clone spanning most of the BMP6 gene hybridized to chromosome 6 in band region p23-p24 using FISH analysis, placing this gene cytogenetically more distal than the region of linkage for CCD. We derived a new polymorphic marker from this same P1 clone and found recombinations between the marker and CCD in this family. The results confirm the map position of CCD on 6p21, further refine the CCD genetic interval by identifying a recombination between D6S451 and D6S459, and exclude BMP6 as a candidate gene.

Concerning the primary defect leading to the pleiotropic effects caused by anophthalmic white (Wh) in the Syrian hamster Mesocricetus auratus.

Ten physiological parameters, controlled by the pituitary-hypothalamic axis, were measured upon males of three genotypes: whwh (cream), Whwh (black eyed white) and WhWh (Anophthalmic white). Univariate analyses of variance comparing data from these hamsters indicated that the gene significantly affects six of these physiological parameters. A principal component and correlation analysis using the multivariate data indicated that of these 10 parameters, six were linearly independent and five were significantly altered by the action of the gene Wh. The results of these analyses and those previously reported suggest that the gene Wh alters the development of the embryonic diencephalon which, in turn, alters the development of the eye and pituitary leading to significant affects on the physiological parameters measured. A new method is described which should, in general, permit the determination of the primary action of a gene in the case of highly pleiotropic mutants.

Effects of pinealectomy on reproduction in the Syrian hamster mutant anophthalmic white (Wh).

The gene Wh, causing anophthalmia in the Syrian hamster, Mesocricetus auratus, is a pleiotropic gene affecting eye development, pigmentation, hearing, and reproduction. Male hamsters homozygous for this gene are usually sterile. Since both Wh and the pineal organ are known to suppress reproductive function, the objective of this study was twofold: (1) to determine whether Wh, by itself, influences testicular differentiation; and (2) to determine whether removal of the pineal gland will restore fertility to both experimentally blinded (B), genetically normal [wh/wh(B)] hamsters and mutant, eyeless (Wh/Wh) hamsters. Accordingly, one testis from each of ten wh/wh(B) and ten Wh/Wh hamsters at approximately 60 days of age was removed, and these testes were compared at the gross and light microscopic level. Since all testes were identical at 60 days of age and contained normal differentiating germ cells, the gene Wh does not appear to affect initial testicular differentiation. Testicular tissues from at least ten wh/wh, wh/wh(B), heterozygous (Wh/wh), and Wh/Wh hamsters, at 135 days of age, were also compared. Testes from all wh/wh(B), and 70% of the Wh/Wh hamsters were hypoplasic and aspermic. Approximately 30% of the testes from Wh/Wh hamsters contained some seminiferous tubules with normal sperm present. Pinealectomy fully restored adult testicular size and morphology in all wh/wh(B) and Wh/Wh hamsters. Thus, it was demonstrated that the atrophy of testes from Wh/Wh individuals is a pineal-mediated phenomenon due to failure of eye development and the subsequent lack of a functional visual pathway. Testes from Wh/Wh hamsters appear to be completely competent to respond to the normal, antigonadotrophic effects of the pineal.

Mouse and hamster mutants as models for Waardenburg syndromes in humans.

Four different Waardenburg syndromes have been defined based upon observed phenotypes. These syndromes are responsible for approximately 2% of subjects with profound congenital hearing loss. At present, Waardenburg syndromes have not been mapped to particular human chromosomes. One or more of the mouse mutant alleles, Ph (patch), s (piebald), Sp (splotch), and Mior (microphthalmia-Oak Ridge) and the hamster mutation Wh (anophthalmic white) may be homologous to mutations causing Waardenburg syndromes. In heterozygotes, phenotypic effects of these four mouse mutations and the hamster mutation are similar to the phenotypes produced by different Waardenburg syndrome mutations. The chromosomal locations and syntenic relationships associated with three of the four mouse mutant genes have been used to predict human chromosomal locations for Waardenburg syndromes: (1) on chromosome 2q near FN1 (fibronectin 1), (2) on chromosome 3p near the proto-oncogene RAF1 or 3q near RHO (rhodopsin), and (3) on chromosome 4p near the proto-oncogene KIT. Waardenburg syndromes show extensive intrafamilial phenotypic variability. Results of our studies with the hamster mutation Wh suggest that this variability may be explained in part by modifier genes segregating within families.

The primary ultrastructural defect caused by anophthalmic white (Wh) in the Syrian hamster.

Anophthalmic white (Wh) of the Syrian hamster is a highly pleiotropic dominant spotting color mutation causing homozygotes to be deaf, blind, and white. An ultrastructural analysis of embryonic tissue reveals that Wh causes the retention of cilia by cells of opposing layers of the embryonic retina and by the lens fiber cells. Previous ultrastructural analyses indicate that Wh also causes the retention of cilia by secretory cells of the anterior lobe of adult pituitaries. We propose that the primary ultrastructural defects caused by Wh is the retention of cilia by embryonic cells. These retained cilia are hypothesized to interfere with normal cell-cell interactions and subsequent cell differentiation.

A histological examination of the pars distalis from the Syrian hamster mutant Anophthalmic white (Wh).

The gene, Wh, causing anophthalmia in the Syrian hamster, Mesocricetus auratus, is a highly pleiotropic gene which has profound effects upon eye development, pigmentation, and reproduction. Hamsters homozygous for this gene also possess abnormalities in testicular size and composition which may be related solely to the lack of the visual pathway acting by means of altered pituitary function. The objective of this study was to determine whether morphological abnormalities existed in hypophyses of male hamsters homozygous for the mutant gene Wh. Accordingly, hypophyses from 10 normal, 10 heterozygous, 10 homozygous mutant, and 5 normal enucleated animals from the AN/As-Wh strain were compared at the light and electron microscopic level. At the light microscopic level, glands from anophthalmic individuals contained 33% fewer cells and many of the cells present either resembled signet ring cells or folliculo stellate cells. At the electron microscopic level, many cells from Wh homozygotes were greatly enlarged, contained only a few organelles and few to no secretory granules. In addition to the glandular cell types, agranular follicular cells were prevalent. Cells with numerous cilia, basal bodies, and a 9 + 2 microtubule configuration were also found within mutant glands. Since pituitaries from hamsters homozygous for Wh displayed far different morphological characteristics than did pituitaries from normal, heterozygous, or normal enucleated animals, it is postulated that either the gene (Wh) acts to alter cellular differentiation of the embryonic hyophysis or that the gene causes abnormal dedifferentiation in the adult.

Waardenburg syndrome (WS): the analysis of a single family with a WS1 mutation showing linkage to RFLP markers on human chromosome 2q.

Waardenburg syndrome type I (WS1; MIM 19350) is caused by a pleiotropic, autosomal dominant mutation with variable penetrance and expressivity. Of individuals with this mutation, 20%-25% are hearing impaired. A multilocus linkage analysis of RFLP data from a single WS1 family with 11 affected individuals indicates that the WS1 mutation in this family is linked to the following four marker loci located on the long arm of chromosome 2: ALPP (alkaline phosphatase, placental), FN1 (fibronectin 1), D2S3 (a unique-copy DNA segment), and COL6A3 (collagen VI, alpha 3). For the RFLP marker loci, a multilocus linkage analysis using MLINK produced a peak lod (Z) of 3.23 for the following linkage relationships and recombination fractions (theta i): (ALPP----.000----FN1)----.122----D2S3----.267----CO L6A3. A similar analysis produced a Z of 6.67 for the following linkage relationships and theta i values among the markers and WS1: (FN1----.000----WS1----.000----ALPP)----.123----D2S 3----.246----COL6A3. The data confirm the conclusion of Foy et al. that at least some WS1 mutations map to chromosome 2q.

Effects of the gene Wh on reproduction in the Syrian hamster, Mesocricetus auratus.

The gene Wh, causing anophthalmia in the Syrian hamster, Mesocricetus auratus, is a highly pleiotropic gene, which has profound effects upon eye development, pigmentation, and reproduction. Since male hamsters homozygous for this gene are usually sterile, the objective of this study was to determine whether the testes of mutant hamsters differed from the normal phenotype. Accordingly, the testicular tissue from ten normal, ten heterozygous, and ten homozygous mutant animals in the AN/As-Wh strain were compared at the gross, light, and electron microscopic level. Testicular tissue from several mutant animals approached the normal phenotype, due to variable expression of the gene. However, most testes from homozygous mutant hamsters were hypoplastic and aspermic. Abnormalities were observed in Leydig cells, Sertoli cells, and in the developing germ cells. Seminiferous tubules contained germinal epithelium arrested in the early spermatid stage of spermiogenesis, possibly due to premature failure of the Golgi apparatus and subsequent dysgenesis of the acrosome. Since the primary action of the gene is unknown, it was postulated that the gene either acts directly to alter pituitary function, or that the abnormalities in reproduction are due to a failure of eye development and subsequent lack of function of the visual pathway.

Three mutations in the paired homeodomain of PAX3 that cause Waardenburg syndrome type 1.

Genomic DNA from probands of various Waardenburg syndrome (WS) families were PCR-amplified using primers flanking the 8 exons of PAX3. The PCR fragments were screened for sequence variants, and subsequently cycle sequenced. Mutations were detected in exon 6 for 3 probands of WS type 1 families. These mutations all occur in the paired homeodomain DNA-binding motif.

The incidence of deafness is non-randomly distributed among families segregating for Waardenburg syndrome type 1 (WS1).

Waardenburg syndrome (WS) is caused by autosomal dominant mutations, and is characterised by pigmentary anomalies and various defects of neural crest derived tissues. It accounts for over 2% of congenital deafness. WS shows high variability in expressivity within families and differences in penetrance of clinical traits between families. While mutations in the gene PAX3 seem to be responsible for most, if not all, WS type 1, it is still not clear what accounts for the reduced penetrance of deafness. Stochastic events during development may be the factors that determine whether a person with a PAX3 mutation will be congenitally deaf or not. Alternatively, genetic background or non-random environmental factors or both may be significant. We compared the likelihoods for deafness in affected subjects from 24 families with reported PAX3 mutations, and in seven of the families originally described by Waardenburg. We found evidence that stochastic variation alone does not explain the differences in penetrances of deafness among WS families. Our analyses suggest that genetic background in combination with certain PAX3 alleles may be important factors in the aetiology of deafness in WS.

A frameshift mutation in the HuP2 paired domain of the probable human homolog of murine Pax-3 is responsible for Waardenburg syndrome type 1 in an Indonesian family.

Waardenburg syndrome type 1 (WS1) is an autosomal dominant disorder characterized by deafness, dystopia canthorum, heterochromia iridis, white forelock, and premature greying. A similar phenotype is caused in the mouse by mutations in the Pax-3 gene. This observation, together with comparisons of conserved syntenies in the murine and human genetic maps, suggested that at least some WS1 mutations should occur in HuP2, the probable human homolog of Pax-3. Two mutations in the HuP2 sequence of individuals with WS1 have been reported recently. Both of them occur in the highly conserved paired box region of the gene, which encodes a DNA binding domain. The functional consequences of these mutations are at present speculative. We report here a 14 bp deletion in the paired domain encoded by exon 2 of HuP2 in an Indonesian family segregating for WS1. This frameshift mutation results in a premature termination codon in exon 3. The HuP2 product is a truncated protein lacking most of the paired domain and all of the predicted homeo domain. We propose that the WS1 phenotype in this family is due to loss of function of HuP2 and discuss two mechanisms for the dominant effect of this mutation.

Septo-optic dysplasia and WS1 in the proband of a WS1 family segregating for a novel mutation in PAX3 exon 7.

A four generation family (UoM1) was ascertained with Waardenburg syndrome type 1 (WS1). The proband exhibited both WS1 and septo-optic dysplasia. A G to C transversion was identified in PAX3 exon 7 in four subjects affected with WS1 in this family including the proband. This glutamine to histidine missense mutation at position 391 may also affect splicing. There are over 50 mutations characterised in PAX3 in WS1 patients; however, this is the first example of a WS1 mutation in exon 7 of PAX3.

Missense mutation in the paired domain of PAX3 causes craniofacial-deafness-hand syndrome.

Craniofacial-deafness-hand syndrome (MIM 122880) is inherited as an autosomal dominant mutation characterized by the absence or hypoplasia of the nasal bones, profound sensorineural deafness, a small and short nose with slitlike nares, hypertelorism, short palpebral fissures, and limited movement at the wrist and ulnar deviations of the fingers. In a family of three affected individuals with this syndrome, a mother and two children, a missense mutation (Asn47Lys) in the paired domain of PAX3 was initially detected by SSCP analysis. PCR amplification using an oligonucleotide with a terminal 3'-residue match for the C-to-G transversion in codon 47 showed the presence of this mutation in the DNA from all affected members. The DNA from unaffected members were refractory to PCR amplification with the mutation-specific oligonucleotide but did amplify a control primer pair in the same PCR reaction tube. A previously described missense mutation in this same codon (Asn47His) is associated with Waardenburg syndrome type 3 (Hoth et al., 1993). Substitution of a basic amino acid for asparagine at residue 47, conserved in all known murine Pax and human PAX genes, appears to have a more drastic effect on the phenotype than missense, frameshift and deletion mutations of PAX3 that cause Waardenburg syndrome type 1.

Effects of Pax3 modifier genes on craniofacial morphology, pigmentation, and viability: a murine model of Waardenburg syndrome variation.

Waardenburg syndrome type 1 is caused by mutations in PAX3. Over 50 human PAX3 mutations that lead to hearing, craniofacial, limb, and pigmentation anomalies have been identified. A PAX3 mutant allele, segregating in a family, can show reduced penetrance and variable expressivity that cannot be explained by the nature of the mutation alone. The Mus musculus Pax3 mutation Spd (Splotch-delayed, Pax3Spd), coisogenic on the C57BL/6J (B6) genetic background, produces in heterozygotes a white belly spot with 100% penetrance and very few other anomalies. By contrast, many Spd/+ BC1 progeny [F1 female Spd/+ (female Spd/+ B6 x male +/+ Mus spretus) x male +/+ B6] exhibit highly variable craniofacial and pigmentary anomalies. Of the BC1 Spd/+ progeny, 23.9% are estimated to be nonviable, and 32.1% are nonpenetrant for the white belly spot. The penetrance and expressivity of the Spd/+ genotype are controlled in part by the genetic background and the sex of the individual. A minimum of two genes interact with Spd to influence the craniofacial features of these mice. One of these genes may be either X-linked or sex-influenced, while the other is autosomal. The A-locus (Agouti) or a gene closely linked to A also plays a role in determining craniofacial features. At least one additional gene, possibly the A-locus or a gene linked to A, interacts with Spd and determines the presence and size of the white belly spot. The viability of BC1 mice is influenced by at least three factors: Spd, A-locus alleles or a gene closely linked to the A-locus, and the sex of the mouse. These BC1 mice provide an opportunity to identify genes that interact with and modify the expression of Pax3 and serve as a model to identify the genes that modify the expression of human PAX3 mutations.