Mutations in the TRKA/NGF receptor gene in patients with congenital insensitivity to pain with anhidrosis.

Congenital insensitivity to pain with anhidrosis (CIPA; MIM 256800) is an autosomal-recessive disorder characterized by recurrent episodes of unexplained fever, anhidrosis (absence of sweating) and absence of reaction to noxious stimuli, self-mutilating behaviour and mental retardation. The genetic basis for CIPA is unknown. Nerve growth factor (NGF) induces neurite outgrowth and promotes survival of embryonic sensory and sympathetic neurons. Mice lacking the gene for TrkA, a receptor tyrosine kinase for NGF, share dramatic phenotypic features of CIPA, including loss of responses to painful stimuli, although anhidrosis is not apparent in these animals. We therefore considered the human TRKA homologue as a candidate for the CIPA gene. The mRNA and genomic DNA encoding TRKA were analysed in three unrelated CIPA patients who had consanguineous parents. We detected a deletion-, splice- and missense-mutation in the tyrosine kinase domain in these three patients. Our findings strongly suggest that defects in TRKA cause CIPA and that the NGF-TRKA system has a crucial role in the development and function of the nociceptive reception as well as establishment of thermoregulation via sweating in humans. These results also implicate genes encoding other TRK and neurotrophin family members as candidates for developmental defect(s) of the nervous system.

Novel and recurrent TRPV4 mutations and their association with distinct phenotypes within the TRPV4 dysplasia family.

BACKGROUND: Mutations in TRPV4, a gene that encodes a Ca(2+) permeable non-selective cation channel, have recently been found in a spectrum of skeletal dysplasias that includes brachyolmia, spondylometaphyseal dysplasia, Kozlowski type (SMDK) and metatropic dysplasia (MD). Only a total of seven missense mutations were detected, however. The full spectrum of TRPV4 mutations and their phenotypes remained unclear. OBJECTIVES AND METHODS: To examine TRPV4 mutation spectrum and phenotype-genotype association, we searched for TRPV4 mutations by PCR-direct sequencing from genomic DNA in 22 MD and 20 SMDK probands. RESULTS: TRPV4 mutations were found in all but one MD subject. In total, 19 different heterozygous mutations were identified in 41 subjects; two were recurrent and 17 were novel. In MD, a recurrent P799L mutation was identified in nine subjects, as well as 10 novel mutations including F471del, the first deletion mutation of TRPV4. In SMDK, a recurrent R594H mutation was identified in 12 subjects and seven novel mutations. An association between the position of mutations and the disease phenotype was also observed. Thus, P799 in exon 15 is a hot codon for MD mutations, as four different amino acid substitutions have been observed at this codon; while R594 in exon 11 is a hotspot for SMDK mutations. CONCLUSION: The TRPV4 mutation spectrum in MD and SMDK, which showed genotype-phenotype correlation and potential functional significance of mutations that are non-randomly distributed over the gene, was presented in this study. The results would help diagnostic laboratories establish efficient screening strategies for genetic diagnosis of the TRPV4 dysplasia family diseases.

Dominant-negative mutations of the tumor suppressor p53 relating to early onset of glioblastoma multiforme.

Previous experiments have suggested that some mutant forms of p53 are able to inactivate the endogenous wild-type p53 protein in a dominant-negative fashion. However, it remains unknown whether tumors with such dominant-negative (transdominant) p53 mutants have a biological significance that is different from that of recessive p53 mutants. In this study, we examined the dominant-negative potential of various p53 mutants using a yeast-based assay in which both wild-type and mutant p53 were efficiently expressed. We tested a total of 106 p53 mutants, which were identified in brain tumors, glioblastoma multiforme-derived cell lines, breast cancers, or premalignant lesions and squamous cell carcinomas of oral epithelium or were otherwise created by mutagenesis. In agreement with the previous studies, our results demonstrated that transdominant mutations affected amino acid residues that are essential for the stabilization of the DNA-binding surface in the p53 core domain and for the direct interaction of p53 with its DNA-binding sequence. Among 40 patients with sporadic glioblastomas, the average age at diagnosis was significantly younger in the patients with tumors harboring dominant-negative mutations (30.4 +/- 14.7 years, n = 7) than it was in those with recessive mutations (55.2 +/- 18.6 years, n = 9, P < 0.012) and in those without mutations (54.7 +/- 17.1 years, n = 24, P < 0.003). Our data suggest that dominant-negative p53 mutants accelerate development and/or growth of glioblastoma anlagen.

High frequency of p53 mutations in human oral epithelial dysplasia and primary squamous cell carcinoma detected by yeast functional assay.

To determine the timing and actual incidence of p53 mutations in oral epithelial lesions, we examined 33 primary squamous cell carcinomas (SCCs), 14 dysplasias and six hyperplasias from Japanese patients by a combination of yeast functional assay and DNA sequencing. The assay detects mutations of p53 mRNA between codons 67 and 347 on the basis of the DNA-binding activity of the protein. Twenty-six SCCs (79%) and five dysplasias (36%) were positive for p53 mutation, while all six hyperplasias were negative for the mutation. Human papillomavirus type 16 E6 mRNA was detected in one of seven p53 mutation-negative SCCs by reverse transcription polymerase chain reaction (RT-PCR). We further examined p53 mutations in 17 Sri Lankan oral SCCs using the yeast functional assay and the single-strand conformation polymorphism analysis of PCR-amplified DNA fragments (PCR-SSCP) of exon 5-8. The mutations were confirmed by DNA sequencing and the detection sensitivity was compared between the two methods. Six samples (35%) were positive for p53 mutation in PCR-SSCP analysis, while nine samples (53%) were positive in yeast functional assay. This suggests that the incidence of p53 mutations has been considerably underestimated in the conventional SSCP analysis. The present data indicate that p53 mutations are extremely frequent in oral cancers in the Japanese, and suggest that the timing and significance of p53 mutation in oral tumor progression vary in different ethnic populations and areas.

Induction of apoptosis in melanoma cell lines by p53 and its related proteins.

Melanoma cells rarely contain mutant p53 and hardly undergo apoptosis by wild-type p53. By using recombinant adenoviruses that express p53 or p53-related p51A or p73beta, we tested their apoptotic activities in melanoma cells. Yeast functional assay revealed a mutation of p53 at the 258th codon (AAA [K] instead of GAA [E]) in one cell line, 70W, out of six human melanoma cell lines analyzed (SK-mel-23, SK-mel-24, SK-mel-118, TXM18, 70W, and G361). Adenovirus-mediated transfer of p53, p51A, and/or p73beta suppressed growth and induced apoptotic DNA fragmentation of SK-mel-23, SK-mel-118, and 70W cells. Interestingly, p51A induced DNA fragmentation in them more significantly than p53 and p73beta. By Western blotting we analyzed levels of apoptosis-related proteins in cells expressing p53 family members. Apoptotic Bax and antiapoptotic Bcl-2 were not significantly upregulated or downregulated by expression of p53, p51A, or p73beta, except for p53-expressing 70W cells, which contained a larger amount of Bax protein than LacZ-expressing cells. Activation of caspase-3 was demonstrated only in p51A-expressing SK-mel-118 cells. We show here that p51A can mediate apoptosis in both wild-type and mutant p53-expressing melanoma cells more significantly than p53 and p73beta. It is also suggested that in melanoma cells (i) cellular target protein(s) other than Bcl-2 and Bax might be responsible for induction of p51A-mediated apoptosis and (ii) caspase-3 is not always involved in the apoptosis by p53 family members.

A new syndrome of dwarfism, brachydactyly, nail dysplasia, and mental retardation in sibs.

We describe a new multiple congenital anomaly/mental retardation (MCA/MR) syndrome in chromosomally normal sibs. Both had microcephaly, a "coarse" face with synophrys, ear anomalies, type B brachydactyly, nail dysplasia, skeletal anomalies, dwarfism, and mental retardation. Their mother had nail dysplasia, mild mental retardation, and short stature. An uncle, a younger brother of the mother, died at 17 years of age and also had a "coarse" face, digital anomalies, dwarfism, and severe mental retardation. The malformation complex in this family apparently has not been described previously, and the manifestations of the patients do not correspond to those of any known malformation syndrome. The disorder may be attributable to the pleiotropic effect of an autosomal dominant or an X-linked semidominant gene.

Cryptomicrotia and short, stubby fingers with excess fingertip arch patterns in a mother and son.

We describe a malformation syndrome of bilateral cryptomicrotia, brachytelomesophalangy, hypoplastic toe nails, and excess fingertip arch patterns in a chromosomally and mentally normal mother and son. Familial occurrence of this malformation complex has not been described previously and the manifestations of the patients do not correspond to those of any known malformation syndromes. The disorder in this family may be attributable to the pleiotropic effect of an autosomal or an X-linked dominant gene.

Patient with del(12)(q12q13.12) manifesting abnormalities compatible with Noonan syndrome.

We report on a Japanese boy with interstitial deletion of chromosome 12q12-q13.12, who had multiple congenital anomalies with severe psychomotor retardation. Most of the clinical manifestations were compatible with Noonan syndrome phenotype except for the absence of cardiac defects. Severe mental retardation and intrauterine onset of growth retardation may have been due to the chromosomal deletion. The interstitial deletion does not overlap a putative Noonan syndrome locus, which was recently assigned to 12q22-qter by linkage analysis. Although correlation between the phenotype and del(12)(q12q13.12) was not confirmed, because this is the first report of deletion of proximal 12q, the deleted segment may contain another Noonan syndrome locus.

Mutchinick syndrome in a Japanese girl.

We report on a 7-year-old Japanese girl with Mutchinick syndrome, a rare congenital malformation syndrome described in a pair of Argentinean sisters and a pair of German brothers; both originating from the same geographic region in the former East Prussia. The girl we describe had most of the clinical manifestations of the syndrome, including growth and developmental retardation, and craniofacial anomalies with microcephaly, hypertelorism, a broad straight nose, low-set malformed ears, and a wide, tented mouth. She also had the following hitherto undescribed manifestations: ventricular septal defect, palmoplantar hyperkeratosis, bilateral partial soft-tissue syndactyly of second and third toes, and megaloureters. The occurrence of the syndrome in a Japanese girl indicates that the syndrome is not restricted to the descendants of individuals from a confined region in northeastern Europe.

Waardenburg syndrome type I in a child with de novo inversion (2)(q35q37.3).

We report on a child with Waardenburg syndrome type I and a paracentric inversion of chromosome 2. This 20 month-old boy has dystopia canthorum, sensorineural deafness, heterochromia iridis, partially albinotic ocular fundi, and partial leukodermia. He does not have mental retardation or any skeletal abnormalities. Family history was unremarkable. Cytogenetic studies demonstrated that the patient has a paracentric inversion (2)(q35q37.3); his parents have normal chromosomes. These findings suggest that the locus of the gene for Waardenburg syndrome type I may be at 2q35 or 2q37.3.

4q33-qter deletion and absorptive hypercalciuria: report of two unrelated girls.

We report on two unrelated girls with multiple malformations, each of whom had a der(4)t(4;?)(q33;?) chromosome--an unbalanced translocation chromosome with deletion of the 4q33-qter segment and addition of a segment of an unknown chromosome. One of the two girls had asymptomatic kidney stones. Both had excess urinary calcium excretion (0.53 and 0.84 mg/mg creatinine, respectively), exaggerated excretion on oral calcium load, and reduced but excessive excretion on restricted calcium intake. The urinary calcium excretion of their parents was normal. Both girls were thus diagnosed to have sporadic absorptive hypercalciuria. It was deduced that the 4q33-qter segment contains the putative gene for absorptive hypercalciuria.

Kabuki make-up (Niikawa-Kuroki) syndrome: a study of 62 patients.

These 62 patients with the Kabuki make-up syndrome (KMS) were collected in a collaborative study among 33 institutions and analyzed clinically, cytogenetically, and epidemiologically to delineate the phenotypic spectrum of KMS and to learn about its cause. Among various manifestations observed, most patients had the following five cardinal manifestations: 1) a peculiar face (100%) characterized by eversion of the lower lateral eyelid; arched eyebrows, with sparse or dispersed lateral one-third; a depressed nasal tip; and prominent ears; 2) skeletal anomalies (92%), including brachydactyly V and a deformed spinal column, with or without sagittal cleft vertebrae; 3) dermatoglyphic abnormalities (93%), including increased digital ulnar loop and hypothenar loop patterns, absence of the digital triradius c and/or d, and presence of fingertip pads; 4) mild to moderate mental retardation (92%); and 5) postnatal growth deficiency (83%). Thus the core of the phenotypic spectrum of KMS is rather narrow and clearly defined. Many other inconsistent anomalies were observed. Important among them were early breast development in infant girls (23%), and congenital heart defects (31%), such as a single ventricle with a common atrium, ventricular septal defect, atrial septal defect, tetralogy of Fallot, coarctation of aorta, patent ductus arteriosus, aneurysm of aorta, transposition of great vessels, and right bundle branch block. Of the 62 KMS patients, 58 were Japanese, an indication that the syndrome is fairly common in Japan. It was estimated that its prevalence in Japanese newborn infants is 1/32,000. All the KMS cases in this study were sporadic, the sex ratio was even, there was no correlation with birth order, the consanguinity rate among the parents was not high, and no incriminated agent was found that was taken by the mothers during early pregnancy. Three of the 62 patients had a Y chromosome abnormality involving a possible common breakpoint (Yp11.2). This could indicate another possibility, i.e., that the KMS gene is on Yp11.2 and that the disease is pseudoautosomal dominant. These findings are compatible with an autosomal dominant disorder in which every patient represents a fresh mutation. The mutation rate was calculated at 15.6 X 10(6).

Molecular study of the Prader-Willi syndrome: deletion, RFLP, and phenotype analyses of 50 patients.

Deletion and RFLP studies with 5 cloned DNA markers localized at 15q11.2 were performed in 50 patients with the Prader-Willi syndrome (PWS). A one-copy density (deletion) for at least one of 4 loci, D15S9, D15S11, D15S10, D15S12, was detected in 32 (64%) of the 50 patients; deletions of each of the 4 loci were found in 29, 30, 29, and 28 patients, respectively. Three patients showed 4 or more copy density for D15S12 locus, in addition to deletions. The remaining 18 patients showed two-copy densities for each of the 4 loci. A common site of rearrangements among our 32 patients as well as the reported patients seemed to be confined to a segment between D15S9 and D15S11, suggesting the putative PWS gene locus in this segment. Of 6 patients who have cytologic deletions but did not show any molecular deletions, 3 have normal size of hands and feet, and 4 have normally pigmented skin and hair. The normal pigmentation was also observed in 3 patients who had small molecular deletions in the examined 5-locus segment. These observations may support the conception of contiguous gene syndrome. RFLP analysis demonstrated maternal uniparental isodisomy of chromosomes 15 in both a patient with 45,t(15q;15q) and a karyotypically normal patient. Based on the results of the present study, a new model is proposed to explain the occurrence of PWS with a variety of chromosome abnormalities, including partial monosomy, disomy, trisomy, and/or tetrasomy for 15q11.2. The normal development may require an even or more "number ratio" of paternally derived allele(s) to maternally derived allele(s) of the gene(s) localized at 15q11.2, and a disturbance of the ratio would lead to the PWS phenotype.

The Wiedemann-Beckwith syndrome: pedigree studies on five families with evidence for autosomal dominant inheritance with variable expressivity.

We describe 18 individuals from five unrelated families with various manifestations of the Wiedemann-Beckwith syndrome. Pedigree analysis was performed on the 5 families and on another 19 families in the literature, each of which included more than one affected person. The following findings were obtained: 1) the clinical manifestations among the affected individuals were highly variable--those obvious in infancy tended to become less distinct with increasing age; 2) the syndrome was transmitted directly and vertically through three generations in four families, and through two generations in seven families; 3) male-to-male transmission was noted once; 4) the sex ratio in the affected individuals was not significantly different from 1; 5) the segregation ratio of the trait among the sibs of the probands was 0.571 +/- 0.066; 6) the affected + carrier/normal ratio was one among the offspring of the affected individuals and obligate carriers; 7) phenotrance (the expected presence of the trait in a generation) of the syndrome in the sibship of probands was complete, whereas that in earlier generations appeared low. The discrepancy is attributable to the lessening of the clinical features with increasing age as well as to a possibly less aggressive search for abnormalities in older generations. These findings indicate that the syndrome is an autosomal dominant trait with variable expressivity. High-resolution chromosome banding analysis in seven affected individuals and their respective parents showed no abnormalities.

Standard growth curves for Japanese patients with Prader-Willi syndrome.

We constructed the standard growth (length/height and weight) curves for Japanese individuals with Prader-Willi syndrome (PWS). Crude height and weight data were collected from 153 males and 99 females with the syndrome, and the collected data were arranged by a mathematical method to construct the curves. Height growth patterns were quite different between PWS and normal children. Mean height of individuals with the syndrome by puberty is -2 SD for normal children, and it drops off far below -2 SD value after puberty. Final mean height is 141.2 +/- 4.8 cm for females (n = 13) and 147.7 +/- 7.7 cm for males (n = 17), showing 15.8 and 21.9 cm below the average height for normal Japanese girls and boys, respectively. Thus, the degree of shortness is more pronounced in male than in female patients. There was no difference in height between those with chromosome 15q deletion and those without. Mean weight at birth of girls (n = 88) and boys (n = 131) were 2.70 +/- 0.45 Kg and 2.62 +/- 0.47 Kg, respectively. These values were smaller than those for normal neonates (P < 0.05, t-test). The weight of PWS children was under the mean value for normal infants by age 2 years, but gradually increase above the mean values for normal children around ages 2-4 years. Overweight in both males and females becomes obvious during prepuberty. Growth patterns are not different between Japanese and Caucasian children with the syndrome. Short stature is more prominent in boys of both ethnic groups, whereas the degree of overweight appears much more severe in Caucasians.

No evidence of PEG1/MEST gene mutations in Silver-Russell syndrome patients.

Silver-Russell syndrome (SRS) is characterized by prenatal and postnatal growth retardation with morphologic anomalies. Maternal uniparental disomy 7 has been reported in some SRS patients. PEG1/MEST is an imprinted gene on chromosome 7q32 that is expressed only from the paternal allele and is a candidate gene for SRS. To clarify its biological function and role in SRS, we screened PEG1/MEST abnormalities in 15 SRS patients from various standpoints. In the lymphocytes of SRS patients, no aberrant expression patterns of two splice variants (alpha and beta) of PEG1/MEST were detected when they were compared with normal samples. Direct sequence analysis failed to detect any mutations in the PEG1/MEST alpha coding region, and there were no significant mutations in the 5'-flanking upstream region containing the predicted promoter and the highly conserved human/mouse genomic region. Differential methylation patterns of the CpG island for PEG1/MEST alpha were normally maintained and resulted in the same pattern as in the normal control, suggesting that there was no loss of imprinting. These findings suggest that PEG1/MEST can be excluded as a major determinant of SRS.

Transcriptional slippage of p53 gene enhanced by cellular damage in rat liver: monitoring the slippage by a yeast functional assay.

The Long-Evans Cinnamon (LEC) rat is a mutant strain characterized by abnormal copper metabolism and a high incidence of hepatitis and hepatoma. Using a yeast-based assay which scores mutants in p53 gene transcripts as red colonies, we detected frequent mutations in the liver of LEC rats. The majority (50-60%) of these were frameshift mutations caused by the insertion of an extra adenine (A) in the regions containing six consecutive adenines. The rate of A insertion was calculated to be 6.9-9.0% of the total p53 cDNA. Insertions of an extra adenine were found almost exclusively in the mRNA (cDNA), especially in the (A)(6) tract located at the most 5'-side (exon 4) among the three (A)(6) tracts (exons 4, 7, and 8), but rarely in the corresponding sites of genomic DNA. Wild-type p53 cDNA was transcribed in vitro into mRNA with the use of SP6 RNA polymerase and tested by the yeast functional assay. Subsequent sequencing detected A insertions at an overall rate of 1.6% in exons 7 and 8 but none in exon 4. This indicates that the A insertion in the exon 4 (A)(6) tract was an in vivo phenomenon rather than an artifact in reverse transcription or polymerase chain reaction. The percentage of red colonies increased sharply to about 20% of the liver samples in the acute hepatitis stage, and returned to control level of those in the chronic hepatitis stage, and increased again slightly to those in the neoplastic stage. The percentage of red colonies correlated with the serum GOT level (r=0.96, p<0.001) but not with the contents of copper and 8-hydroxydeoxyguanosine in the liver of LEC rats. Ethanol treatment of hepatic cell lines also increased the rate of transcriptional slippage at the (A)(6) tract. These findings indicate that cellular damage is responsible for the increase in the rate of mutation at the transcriptional level, and suggest that cellular damage degrades transcriptional fidelity, thereby further impairing cellular functions.

[Establishment of Chinese hamster ovary cell lines with reduced expression of glutathione reductase after antisense-oriented gene transfection and assessment of the sensitivity to oxidant injury].

Glutathione reductase (GR) protects tissues from oxidant injury by catalysing the reduction of glutathione disulfide (GSSG) to glutathione (GSH). In order to study the effect of GR in protecting cells from oxidant injury, we generated Chinese hamster ovary (CHO) cell lines stably transformed after antisense-oriented gene transfection. The coding region of the human GR was cloned using revere transcription PCR method and selected by transient expression study in mammalian cells. A clone HGR135 showed overexpression of GR in CHO cells and was proved to have no base substitution. This clone, then, was ligated into MEP4 expression vector in an antisense orientation to the human metallothionein promoter and transfected to CHO cells with polybrene. Among 12 cell lines isolated, G17 showed to have the least GR activity (48% of the control), while another four were mildly GR deficient. Southern hybridization of genomic DNA digests and transformation experiment on E. coli revealed that the promoter-antisense coding region component was integrated. Northern hybridization detected reduced amount of GR transcript but no antisense message. Baseline cellular GSH concentrations were lower in G17 than in control (25.7 +/- 2.5 vs. 36.1 +/- 1.9 nmole/mg protein, P < 0.05), while cellular GSSG concentrations were higher (0.61 +/- 0.19 vs. 0.39 +/- 0.09 nmole/mg protein, P < 0.05). After four hours of treatment of G17 and control cells with increasing doses (1 to 10 mM) of t-butylhydroperoxide (t-BuOOH), cellular GSH concentrations in G17 decreased with an elevation of GSSG concentration at 1 mM followed by no further increase at higher t-BuOOH concentration, while GSSG concentrations increased in the control cells without reduction of GSH concentrations at 1-5 mM t-BuOOH treatment. The concentrations of GSH were lower in G17 than in controls at all doses of t-BuOOH. Four hours of exposure to 10 mM t-BuOOH resulted in greater LDH release in G17 than in control (57.3 +/- 4.7 vs. 32.1 +/- 6.5%, P < 0.05). Similarly, G17 cells released more of their LDH to the media than did CHO cells in response to exposure to 95% O2 for 72 hours (19.3 +/- 5.9 vs. 11.9 +/- 5.4%, P < 0.05). The partial GR deficiency in G17 cells impairs their ability to recycle GSSG and this deficiency offers the best explanation for the increased sensitivity of these cells to injury by t-BuOOH or hyperoxia.