OA1 mutations and deletions in X-linked ocular albinism

X-linked ocular albinism (OA1), Nettleship-Falls type, is characterized by decreased ocular pigmentation, foveal hypoplasia, nystagmus, photodysphoria, and reduced visual acuity. Affected males usually demonstrate melanin macroglobules on skin biopsy. We now report results of deletion and mutation screening of the full-length OA1 gene in 29 unrelated North American and Australian X-linked ocular albinism (OA) probands, including five with additional, nonocular phenotypic abnormalities (Schnur et al. 1994). We detected 13 intragenic gene deletions, including 3 of exon 1, 2 of exon 2, 2 of exon 4, and 6 others, which span exons 2-8. Eight new missense mutations were identified, which cluster within exons 1, 2, 3, and 6 in conserved and/or putative transmembrane domains of the protein. There was also a splice acceptor-site mutation, a nonsense mutation, a single base deletion, and a previously reported 17-bp exon 1 deletion. All patients with nonocular phenotypic abnormalities had detectable mutations. In summary, 26 (approximately 90%) of 29 probands had detectable alterations of OA1, thus confirming that OA1 is the major locus for X-linked OA.     

Locus heterogeneity for Waardenburg syndrome is predictive of clinical subtypes

Waardenburg syndrome (WS) is a dominantly inherited and clinically variable syndrome of deafness, pigmentary changes, and distinctive facial features. Clinically, WS type I (WS1) is differentiated from WS type II (WS2) by the high frequency of dystopia canthorum in the family. In some families, WS is caused by mutations in the PAX3 gene on chromosome 2q. We have typed microsatellite markers within and flanking PAX3 in 41 WS1 kindreds and 26 WS2 kindreds in order to estimate the proportion of families with probable mutations in PAX3 and to study the relationship between phenotypic and genotypic heterogeneity. Evaluation of heterogeneity in location scores obtained by multilocus analysis indicated that WS is linked to PAX3 in 60% of all WS families and in 100% of WS1 families. None of the WS2 families were linked. In those families in which equivocal lod scores (between -2 and +1) were found, PAX3 mutations have been identified in 5 of the 15 WS1 families but in none of the 4 WS2 families. Although preliminary studies do not suggest any association between the phenotype and the molecular pathology in 20 families with known PAX3 mutations and in four patients with chromosomal abnormalities in the vicinity of PAX3, the presence of dystopia in multiple family members is a reliable indicator for identifying families likely to have a defect in PAX3.     

Duplication cyst of the antrum: a case report

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.     

Unusual involvement of bone in primary systemic amyloidosis

Vesicoureteric reflux (VUR) is a common childhood condition characterised by regurgitation of urine from the bladder to the kidney. It is the commonest cause of end stage renal failure in children and an important cause in adults. Primary VUR is often familial, suggesting that genetic factors play an important role in its aetiology. Recently, VUR was observed as part of a syndrome, involving optic nerve colobomas and renal anomalies, caused by mutations of the PAX2 gene. PAX2 is a member of the paired box family of genes and is expressed in the ureteric bud and differentiating nephrogenic mesenchyme of the developing kidney. PAX2 has been shown to play a critical role in the development of both the kidney and the ureter. The occurrence of VUR in one family with the PAX2 mutation, and the expression pattern of PAX2 in developing ureteric bud, strongly suggested that PAX2 could be the cause of primary familial VUR. Single strand conformational polymorphism (SSCP) analysis of 23 affected subjects in eight families with primary familial VUR showed no alterations in exons 2-5 of the PAX2 gene. In addition, a polymorphic dinucleotide repeat marker located within the PAX2 gene segregated independently of the disease trait in one large family who primarily had VUR or reflux nephropathy. These results suggest that PAX2 is not a major cause of primary familial reflux.     

PAX genes and human neural tube defects: an amino acid substitution in PAX1 in a patient with spina bifida

From studies in the mouse and from the clinical and molecular analysis of patients with type 1 Waardenburg syndrome, particular members of the PAX gene family are suspected factors in the aetiology of human neural tube defects (NTD). To investigate the role of PAX1, PAX3, PAX7, and PAX9, allelic association studies were performed in 79 sporadic and 38 familial NTD patients from the Dutch population. Sequence variation was studied by SSC analysis of the paired domain regions of the PAX1, PAX7, and PAX9 genes and of the complete PAX3 gene. In one patient with spina bifida, a mutation in the PAX1 gene was detected changing the conserved amino acid Gln to His at position 42 in the paired domain of the protein. The mutation was inherited through the maternal line from the unaffected grandmother and was not detected in 300 controls. In the PAX3 gene, variation was detected at several sites including a Thr/Lys amino acid substitution in exon 6. All alleles were present among patients and controls in about the same frequencies. However, an increased frequency of the rare allele of a silent polymorphism in exon 2 was found in NTD patients, but no significant association was observed (p = 0.06). No sequence variation was observed in the paired domain of the PAX7 and PAX9 genes. Our findings so far do not support a major role of the PAX genes examined in the aetiology of NTD. However, the detection of a mutation in PAX1 suggests that, in principle, this gene can act as a risk factor for human NTD.     

Alport syndrome. A review of the ocular manifestations

Alport syndrome has a prevalence of 1/5000, and 85% of patients have the X-linked form, where affected males develop renal failure and usually have a high-tone sensorineural deafness by the age of 20. The typical ocular associations are a dot-and-fleck retinopathy which occurs in about 85% of affected adult males, anterior lenticonus which occurs in about 25%, and the rare posterior polymorphous corneal dystrophy. The retinopathy and anterior lenticonus are not usually demonstrated in childhood but worsen with time so that the retinal lesion is often present at the onset of renal failure, and the anterior lenticonus, later. The demonstration of a dot-and-fleck retinopathy in any individual with a family history of Alport syndrome or with end-stage renal disease is diagnostic of Alport syndrome. The presence of anterior lenticonus or posterior polymorphous corneal dystrophy in any individual is highly suggestive of the diagnosis of Alport syndrome. Additional ocular features described in X-linked Alport syndrome include other corneal dystrophies, microcornea, arcus, iris atrophy, cataracts, spontaneous lens rupture, spherophakia, posterior lenticonus, a poor macular reflex, fluorescein angiogram hyperfluorescence, electrooculogram and electroretinogram abnormalities, and retinal pigmentation. All mutations demonstrated to date in X-linked Alport syndrome have affected the COL4A5 gene which encodes the alpha 5 chain of type IV collagen. This protein is probably common to the basement membranes of the glomerulus, cochlea, retina, lens capsule, and cornea. However, the alpha 3(IV) and 4(IV) as well as the alpha 5(IV) collagen chains are usually absent from the affected basement membranes, because the abnormal alpha 5(IV) molecule interferes with the stability of all three. The loss of these collagen molecules from the affected basement membranes results in an abnormal ultrastructural appearance. The ocular and other clinical features of autosomal recessive Alport syndrome are identical to those seen in X-linked disease, while retinopathy and cataracts are the only ocular abnormalities described in the rare autosomal dominant form of Alport syndrome. There are no ocular associations of thin basement membrane disease which is a common disease that probably represents the heterozygous expression of X-linked or autosomal recessive Alport syndrome.     

CNS tissue Cu/Zn superoxide dismutase (SOD1) mutations in motor neurone disease (MND)

DNA extracted from CNS tissue of 79 cases of motor neurone disease (MND) was screened by single strand conformation analysis (SSCA) and heteroduplex analysis (HA) for mutations in the Cu/Zn superoxide dismutase (SOD1) gene. The aims were to determine whether somatic mutations of SOD1 may underlie some cases of MND and to characterize the genetic abnormalities by sequencing, for subsequent correlation with the molecular pathological phenotype. In 3 cases a point mutation was found in exon 4: E100G in one familial case, and I113T in two cases (one familial, one sporadic). Two cases had previously undescribed mutations in the 3' untranslated region (3'UTR) of SOD1 and one case had a single base substitution in the intronic sequence upstream from exon 2. None of these patients had a positive family history. Non-CNS tissue was available for 3 out of the 6 cases in whom changes were found. In all 3 the same changes were consistently found in both CNS and non-CNS tissue, excluding the presence of somatic mutations in SOD1. We investigated many MND blood samples and normal controls for the presence of the 3'UTR deletions. We found the 4 bp deletion in 1/90 sporadic MND patients and 1/209 non-MND controls. If the 3'UTR deletions are pathogenic, they would have to operate via a loss of the function mechanism, and further work is necessary to define their significance.     

A novel germline mutation in exon 5 of the multiple endocrine neoplasia type 1 gene

The autosomal dominant multiple endocrine neoplasia type 1 (MEN1) syndrome is characterized by neoplasia of parathyroids, anterior pituitary, and gastrointestinal and pancreatic neuroendocrine tissues. Recently the gene responsible for the MEN1 syndrome has been identified on chromosome region 11q13. Most of the described mutations are nucleotide substitutions and small deletions affecting exons 2 and 3, causing protein truncation. Only one mutation in exon 5 has been found, and this corresponds to a MEN1 sporadic case. Small insertions are also rare. We studied a MENI family composed of five members, two of whom were clinically affected. We found a new germline 1 basepair insertional mutation affecting the exon 5 of the MEN1 gene in the two members affected in this MEN1 family.     

Mutations in exon 7 of the GTP-binding protein Gs alpha were not a common cause of pseudohypoparathyroidism with Albright's hereditary osteodystrophy in Japanese

The identification of unique point mutations in patients with pseudohypoparathyroidism (PHP) with Albright's hereditary osteodystrophy (AHO) in different ethnic backgrounds has proved that defects within the Gs alpha gene account for Gs alpha deficiency in those patients. To search a mutation hot spot of the Gs alpha gene in Japanese patients, we have screened exons 2-13 of the Gs alpha gene for mutations in three unrelated Japanese PHP patients with AHO. We could find no abnormalities by denaturing gradient gel electrophoresis and no mutations of sequencing of exon 7 in these subjects. This suggests that mutations in exon 7 of the Gs alpha gene may not be a common cause of PHP with AHO in Japanese.     

Effect of 5-hydroxytryptamine on bioelectric properties of airway epithelial cells in culture

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.     

Analysis of phenotypic features and FGFR2 mutations in Apert syndrome

A phenotypic and genotypic survey was conducted on 36 Apert syndrome patients. In all but one patient, an FGFR2 mutation, either S252W or P253R, was found in exon IIIa (exon U or 7). The frequency was 71% and 26%, for the mutations S252W and P253R, respectively. These mutations occur in the linker region between immunoglobulin-like domains II and III, which are involved in activation of the receptor by ligand binding and dimerization. The fact that one patient did not have a mutation in the same exon suggests further genetic heterogeneity in Apert syndrome. The frequencies of occurrence or means for measurements of 29 different clinical features (including severity of craniofacial features, syndactyly of the hands and feet, and multisystem involvement) were determined for all patients and for the two subgroups defined by their mutations. Comparison between the subgroups for the different clinical features was performed and suggested no statistically significant differences. These results are not unexpected, because the two common mutations for Apert syndrome alter FGFR2 at adjacent amino acids that are likely to have similar biological, and therefore phenotypic, consequences.     

Molecular characterization of germline mutations in neurofibromatosis 2 in two families

Neurofibromatosis 2 (NF2) is an autosomal dominant disorder that predisposes patients to central nervous system tumors. It is caused by mutations in the NF2 tumor suppressor gene, which is located on chromosome 22q12. We studied 2 multigenerational NF2 families (three members of family 1 and the proband of the family) by gene mutation analysis and clinical assessment. One member of family 1 had a 169 C-->T point mutation at codon 57 of exon 2 and had a severe phenotype. His father had a silent 1113 C-->T point mutation at codon 371 of exon 11 and had a normal phenotype. The proband of family 2 had a deletion at nucleotide 720 G (codon 240) of exon 8. This led to a frameshift and termination at codon 250, and a severe NF2 phenotype. Our results indicate that clinical abnormalities can be present in carriers. Nonsense and frameshift mutations in the NF2 tumor suppressor gene are associated with phenotypes. The clinical abnormalities can develop at a young age.     

De novo alu-element insertions in FGFR2 identify a distinct pathological basis for Apert syndrome

Apert syndrome, one of five craniosynostosis syndromes caused by allelic mutations of fibroblast growth-factor receptor 2 (FGFR2), is characterized by symmetrical bony syndactyly of the hands and feet. We have analyzed 260 unrelated patients, all but 2 of whom have missense mutations in exon 7, which affect a dipeptide in the linker region between the second and third immunoglobulin-like domains. Hence, the molecular mechanism of Apert syndrome is exquisitely specific. FGFR2 mutations in the remaining two patients are distinct in position and nature. Surprisingly, each patient harbors an Alu-element insertion of approximately 360 bp, in one case just upstream of exon 9 and in the other case within exon 9 itself. The insertions are likely to be pathological, because they have arisen de novo; in both cases this occurred on the paternal chromosome. FGFR2 is present in alternatively spliced isoforms characterized by either the IIIb (exon 8) or IIIc (exon 9) domains (keratinocyte growth-factor receptor [KGFR] and bacterially expressed kinase, respectively), which are differentially expressed in mouse limbs on embryonic day 13. Splicing of exon 9 was examined in RNA extracted from fibroblasts and keratinocytes from one patient with an Alu insertion and two patients with Pfeiffer syndrome who had nucleotide substitutions of the exon 9 acceptor splice site. Ectopic expression of KGFR in the fibroblast lines correlated with the severity of limb abnormalities. This provides the first genetic evidence that signaling through KGFR causes syndactyly in Apert syndrome.     

Nonsense mutations in the OCRL-1 gene in patients with the oculocerebrorenal syndrome of Lowe

A candidate gene, OCRL-1, for the oculocerebrorenal syndrome of Lowe (OCRL) has been identified via positional cloning strategies. We have now developed RT-PCR techniques which allow amplification of nearly all of the open reading frame from total RNA and have used the PCR products for mutational analysis. Single strand conformational polymorphism analysis detected aberrant migration in two unrelated patients, both of whom were shown to have the same nonsense mutation at base 2746 on direct sequencing. An additional patient was found to be missing a segment from his RNA that corresponds to an entire exon. The identification of mutations in the OCRL-1 gene provides strong genetic evidence for its being the gene involved in Lowe syndrome.     

Mutations in haemophilia A

In the present study DNA from 281 unrelated haemophilia A patients including 15 inhibitor patients has been analysed by Southern blotting technique. Using various restriction enzymes, cloned factor VIII cDNA probes and genomic fragments we have identified 14 mutations. Six of the mutations are novel partial factor VIII gene deletions. One deletion affects exon 1, two deletions concern exon 6, another deletion, of which breakpoints are sequenced, takes part of exon 16 and two deletions affect exon 26. Besides the deletions, eight point mutations have been found at the TaqI restriction sites of exons 18, 24 and 26. Five C-->T mutations resulted in nonsense mutations, one in exon 18, one in exon 26 and three in exon 24. Two G-->A mutations caused a missense mutation in exon 24 leading to an arginine/glutamine exchange. Although two patients showed this mutation, their clinical phenotypes were different, possibly due to an additional unidentified sequence polymorphism. A G-->T mutation in exon 26 substituted the arginine with leucine. All deletions and seven of the point mutations are associated with severe disease with a detectable inhibitor in the patient with the TaqI-point mutation in exon 18. One of the G-->A mutations is associated with mild haemophilia but the patient also has developed an inhibitor. Amongst these mutations the origin of the mutation could be determined in four kindred, one of which showed maternal mosaicism.     

Carbonic anhydrase II deficiency syndrome--clinico-pathological, biochemical and molecular studies

We reported on three unrelated Japanese families with carbonic anhydrase II (CA II) deficiency syndrome. In the present study, the CA II gene was sequenced in the family of a patient with hybrid type renal tubular acidosis whose parents were nonconsanguineous, and a T to G transition at exon 2 was identified. The change results in the substitution of the stop codon (TAG) at position 40 for Tyr (TAT). The maternal and paternal mutations were the same suggesting that they were obligate heterozygotes. This is a novel mutation in the CA II deficiency syndrome, which has not been described before.     

Evolution of precipitates in lead-implanted aluminum: A backscattering and channeling study

BACKGROUND: Thirty-six mutations that cause Gaucher disease, the most common glycolipid storage disorder, are known. Although both alleles of most patients with the disease contain one of these mutations, in a few patients one or both disease-producing alleles have remained unidentified. Identification of mutations in these patients is useful for genetic counseling. MATERIALS AND METHODS: The DNA from 23 Gaucher disease patients in whom at least one glucocerebrosidase allele did not contain any of the 36 previously described mutations has been examined by single strand conformation polymorphism (SSCP) analysis, followed by sequencing of regions in which abnormalities were detected. RESULTS: Eight previously undescribed mutations were detected. In exon 3, a deletion of a cytosine at cDNA nt 203 was found. In exon 6, three missense mutations were identified: a C-->A transversion at cDNA nt 644 (Ala176-->Asp), a C-->A transversion at cDNA nt 661 that resulted in a (Pro182-->Thr), and a G-->A transition at cDNA nt 721 (Gly202-->Arg). Two missense mutations were found in exon 7: a G-->A transition at cDNA nt 887 (Arg257-->Gln) and a C-->T at cDNA nt 970 (Arg285-->Cys). Two missense mutations were found in exon 9: a T-->G at cDNA nt 1249 (Trp378-->Gly) and a G-->A at cDNA nt 1255 (Asp380-->Asn). In addition to these disease-producing mutations, a silent C-->G transversion at cDNA nt 1431, occurring in a gene that already contained the 1226G mutation, was found in one family. CONCLUSIONS: The mutations described here and previously known can be classified as mild, severe, or lethal, on the basis of their effect on enzyme production and on clinical phenotype, and as polymorphic or sporadic, on the basis of the haplotype in which they are found. Rare mutations such as the new ones described here are sporadic in nature.