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.

SummaryNerve growth factor (NGF) induces neurite outgrowth and promotes survival of embryonic sensory and sympathetic neurons. TRKA, a receptor tyrosine kinase cloned from a human colon cancer was later found to be expressed in the nervous system and phosphorylated in response to NGF. Somatic rearrangement(s) of the TRKA gene (also designated NTRK1) are responsible for formation of some oncogenes. Genetic defects in TRKA are responsible for a human disorder, congenital insensitivity to pain with anhidrosis (CIPA). We report here isolation and characterization of the TRKA gene which spans at least 23 kb and is split into 17 exons. Exon sizes range from 18 to 394 bp and intron sizes range from 170 bp to at least 3.3 kb. Sizes and boundaries of the exons were determined, and all the splice donor and acceptor sites conformed to the GT/AG rule. Approximately 1.2 kb of the 5'-flanking regions was sequenced, and putative regulatory elements were identified. These results will be useful for studies on the developmental and biological regulation of the TRKA gene and for further characterization of mutations in CIPA patients as well as elucidation of mechanisms responsible for rearrangement(s) observed in human tumors.

Familial idiopathic low-molecular-weight proteinuria (FILMWP) is a renal proximal tubulopathy that occurs predominantly in males. FILMWP is characterized by mild proteinuria consisting of low-molecular-weight proteinuria, aminoaciduria and relatively conserved renal function, but without rickets. To determine whether FILMWP is related to the CLCN5 gene, which is responsible for Dent's disease and two related disorders, we analyzed the CLCN5 gene from four Japanese families with FILMWP. We identified two novel mutations: one was a single base insertion at codon 520 serine in exon 10 and the other was a single base deletion at codon 403 tyrosine in exon 8. These mutations caused a shift in the reading frame, resulting in synthesis of truncated CLC5 proteins that lacked 220 (29%) and 314 (42%) amino acids, respectively. These mutations were demonstrated to cosegregate with the disease in two families, respectively. We conclude that the CLCN5 gene is responsible for this proximal renal tubulopathy in some Japanese families and that FILMWP is possibly a variant of Dent's disease.

The E2 gene of the branched-chain α-keto acid dehydrogenase (BCKDH) complex was studied at the molecular level in three patients with intermittent maple syrup urine disease (MSUD). All three patients had higher BCKDH activity than did those with the classical phenotype. In the first patient, a single base substitution from A to G in intron 8 created a new 5Ј splice site and caused an insertion of 126 nucleotides between exons 8 and 9 by activating an upstream cryptic 3Ј splice site in the same intron. The predicted mRNA encoded a truncated protein with 282 amino acids including 4 novel ones at the carboxyl terminus, compared with the normal protein with 421 amino acids. In vitro, the region from the patient but not from a normal control was recognized and was recovered as a novel exon, indicating that the single substitution was responsible for incorporation of the region into mRNA. This mutation probably supports an exon definition model in which the spliceosome recognizes a 3Ј splice site and then scans downstream for an acceptable 5Ј splice site, thereby defining an exon. The second patient was homozygous for a G to T transversion at nucleotide 1463 in exon 11, which predicted a substitution of the termination codon by a leucine residue and the addition of 7 extra amino acids at the carboxyl terminus. For each mutation, these two patients were homozygous and their parents were heterozygous. The third patient was a compound heterozygote for a C to G transversion at nucleotide 309 in exon 4 and a G to A transition at nucleotide 1165 in exon 9, causing an Ile-toMet substitution at amino acid 37 and a Gly-to-Ser substitution at amino acid 323, respectively. Taken together, these results indicate that the molecular basis of intermittent phenotype MSUD in some patients can be due to mutations in the E2 gene, giving rise to a low but significant residual activity of the BCKDH complex.

Mast cells (MCs) are crucial cells in the development of various types of diseases, including allergy, atopic dermatitis and hair disorders. Corticotropin releasing hormone (CRH) plays a central role in the stress response by regulating the hypothalamic-pituitary-adrenal (HPA) axis. However, CRH and its receptors (CRHR) have been recently identified within the skin and reported to play important roles in local cutaneous HPA axis. MCs are also reported to act as CRH mediated stress sentinels in tissue. We have previously shown that CRH induces connective tissue type (CT)-MC degranulation and promotes maturation. Here, we investigated whether CRH can also affect mucosal type (M)-MC biology by using human nasal polyp organ culture. The expression of both CRH and CRHR was detected within nasal polyp samples by immunohistochemistry and RT-PCR. CRH treatment in the organ culture increased the expression of ACTH receptor as well as glucocorticosteroid receptor in situ. Kit or tryp-tase+ M-MCs of isolated nasal polyps was shown to express CRHR by double immunofluorescence. CRH treatment significantly increased tryptase+ M-MC number and induced degranulation in situ. This was partially abrogated by the co-administration of stem cell factor (SCF) neutralizing antibody and CRHR antagonist, antalarmin. Just as human hair follicles organ culture, CRH also increased SCF expression within the epithelium of organ cultured nasal polyp samples. In contrast to CT-MC, CRH treatment significantly increased tryptase+ M-MC proliferation in situ. Furthermore, the effect of CRH on M-MCs degranulation and number was partially diminished by CRHR gene knockdown in situ. These results indicate that CRH induces both cutaneous and mucosal type neuroinflammation by promoting MC activity and increasing the number of it and that blockade of SCF and CRHR might be a novel future target for treating stress-induced disorders including atopic dermatitis and asthma.