Mutations in NALP12 cause hereditary periodic fever syndromes.

NALP proteins, also known as NLRPs, belong to the CATERPILLER protein family involved, like Toll-like receptors, in the recognition of microbial molecules and the subsequent activation of inflammatory and immune responses. Current advances in the function of NALPs support the recently proposed model of a disease continuum bridging autoimmune and autoinflammatory disorders. Among these diseases, hereditary periodic fevers (HPFs) are Mendelian disorders associated with sequence variations in very few genes; these variations are mostly missense mutations whose deleterious effect, which is particularly difficult to assess, is often questionable. The growing number of identified sporadic cases of periodic fever syndrome, together with the lack of discriminatory clinical criteria, has greatly hampered the identification of new disease-causing genes, a step that is, however, essential for appropriate management of these disorders. Using a candidate gene approach, we identified nonambiguous mutations in NALP12 (i.e., nonsense and splice site) in two families with periodic fever syndromes. As shown by means of functional studies, these two NALP12 mutations have a deleterious effect on NF-kappaB signaling. Overall, these data identify a group of HPFs defined by molecular defects in NALP12, opening up new ways to manage these disorders. The identification of these first NALP12 mutations in patients with autoinflammatory disorder also clearly demonstrates the crucial role of NALP12 in inflammatory signaling pathways, thereby assigning a precise function to this particular member of an emerging family of proteins whose putative biological properties are currently inferred essentially through in vitro means.

PYPAF1 nonsense mutation in a patient with an unusual autoinflammatory syndrome: role of PYPAF1 in inflammation.

OBJECTIVE: To gain insight into the pathophysiology of an unusual autoinflammatory syndrome, in a patient of Armenian origin, that mimicked familial Mediterranean fever (FMF) but with episodes triggered by generalized exposure to cold, and to further elucidate the controversial function of the protein encoded by PYPAF1, whose mutations (exclusively missense to date) have been identified in 3 hereditary recurrent fever syndromes. METHODS: The patient's DNA was screened for mutations in both MEFV, the gene responsible for FMF, and PYPAF1. The ability of different recombinant PYPAF1 isoforms, expressed in HEK 293 cells, to regulate NF-kappaB signaling was subsequently assessed. RESULTS: No disease-causing mutation was found in MEFV. However, a nonsense mutation (p.Arg554X) was identified in PYPAF1; this defect resulted in a truncated protein lacking all leucine-rich repeats. Study of the wild-type and mutant PYPAF1 recombinant proteins revealed that PYPAF1 inhibited NF-kappaB proinflammatory pathways, and that the identified nonsense mutation impaired this property. CONCLUSION: These molecular and clinical findings, together with the clinical manifestations in the patient, which call into question the current nosology of the hereditary recurrent fever syndromes, are consistent with the hypothesis that PYPAF1 acts as an inhibitor of NF-kappaB signaling. They also provide a clear elucidation of the functional consequences of this nonsense PYPAF1 mutation not previously described in the literature, which result in a partial loss of function and may thereby explain the pathophysiology of the autoinflammatory syndrome observed in this patient.

Syndromic short stature in patients with a germline mutation in the LIM homeobox LHX4.

Studies of genetically engineered flies and mice have revealed the role that orthologs of the human LIM homeobox LHX4 have in the control of motor-neuron-identity assignment and in pituitary development. Remarkably, these mouse strains, which bear a targeted modification of Lhx4 in the heterozygous state, are asymptomatic, whereas homozygous animals die shortly after birth. Nevertheless, we have isolated the human LHX4 gene, as well as the corresponding cDNA sequence, to test whether it could be involved in developmental defects of the human pituitary region. LHX4, which encodes a protein 99% identical to its murine counterpart, consists of six coding exons and spans >45 kb of the q25 region of chromosome 1. We report a family with an LHX4 germline splice-site mutation that results in a disease phenotype characterized by short stature and by pituitary and hindbrain (i.e., cerebellar) defects in combination with abnormalities of the sella turcica of the central skull base. This intronic mutation, which segregates in a dominant and fully penetrant manner over three generations, abolishes normal LHX4 splicing and activates two exonic cryptic splice sites, thereby predicting two different proteins deleted in their homeodomain sequence. These findings, which elucidate the molecular basis of a complex Mendelian disorder, reveal the fundamental pleiotropic role played by a single factor that tightly coordinates brain development and skull shaping during head morphogenesis.

Alternative splicing at the MEFV locus involved in familial Mediterranean fever regulates translocation of the marenostrin/pyrin protein to the nucleus.

Mutations in MEFV, a gene encoding a protein (marenostrin/pyrin) of unknown function, are associated with familial Mediterranean fever, a genetic condition characterized by febrile episodes of serosal inflammation. Based on its primary structure, this 781 residue protein is thought to function as a nuclear effector molecule. However, recent transient expression studies indicated a perinuclear cytoplasmic localization. Here, we describe the isolation and expression of a novel human MEFV isoform, MEFV-d2, generated by in-frame alternative splicing of exon 2. This transcript, expressed in leukocytes, predicts a 570 residue protein designated marenostrin-d2. To investigate differences in subcellular localization between the full-length protein (marenostrin-fl) and marenostrin-d2, while providing against the overexpression of transiently expressed proteins, we have generated CHO cell lines stably expressing these two isoforms fused to the green fluorescent protein. The localization pattern of marenostrin-d2 differs dramatically from that of marenostrin-fl. Marenostrin-fl is homogeneously distributed over the entire cytoplasm, whereas marenostrin-d2 concentrates into the nucleus. To map the critical domain(s) specifying these differences, deletion mutants have been generated. Deletion of the putative nuclear localization signals (NLS) does not alter the nuclear localization of marenostrin-d2 whereas, despite the lack of discernible NLS in the domain encoded by the exon 1-exon 3 splice junction, deletion of this domain indeed disrupts this localization. These data, which challenge the current domain organization model of marenostrin, strongly suggest that MEFV encodes a nuclear protein and raises the possibility that MEFV alternative splicing may control functions of wild-type and mutant marenostrin proteins by regulating their translocation to the nucleus.

Identification of MEFV-independent modifying genetic factors for familial Mediterranean fever.

Familial Mediterranean fever (FMF) is a recessively inherited disorder predisposing to renal amyloidosis and associated with mutations in MEFV, a gene encoding a protein of unknown function. Differences in clinical expression have been attributed to MEFV-allelic heterogeneity, with the M694V/M694V genotype associated with a high prevalence of renal amyloidosis. However, the variable risk for patients with identical MEFV mutations to develop this severe complication, prevented by lifelong administration of colchicine, strongly suggests a role for other genetic and/or environmental factors. To overcome the well-known difficulties in the identification of modifying genetic factors, we investigated a relatively homogeneous population sample consisting of 137 Armenian patients with FMF from 127 independent families living in Armenia. We selected the SAA1, SAA2, and APOE genes-encoding serum amyloid proteins and apolipoprotein E, respectively-as well as the patients' sex, as candidate modifiers for renal amyloidosis. A stepwise logistic-regression analysis showed that the SAA1alpha/alpha genotype was associated with a sevenfold increased risk for renal amyloidosis, compared with other SAA1 genotypes (odds ratio [OR] 6. 9; 95% confidence interval [CI] 2.5-19.0). This association, which was present whatever the MEFV genotype, was extremely marked in patients homozygous for M694V (11/11). The risk for male patients of developing renal amyloidosis was fourfold higher than that for female patients (OR=4.0; 95% CI=1.5-10.8). This association, particularly marked in patients who were not homozygous for M694V (34.0% vs. 11.6%), was independent of SAA1-allelic variations. Polymorphisms in the SAA2 or APOE gene did not appear to influence susceptibility to renal amyloidosis. Overall, these data, which provide new insights into the pathophysiology of FMF, demonstrate that susceptibility to renal amyloidosis in this Mendelian disorder is influenced by at least two MEFV-independent factors of genetic origin-SAA1 and sex-that act independently of each other.

Species-specific alternative splice mimicry at the growth hormone receptor locus revealed by the lineage of retroelements during primate evolution.

In humans, growth hormone receptor (GHR) transcripts exist in two isoforms differing by the retention (GHRfl) or exclusion (GHRd3) of exon 3, whereas in mice GHRfl is solely expressed. This species-specific expression pattern is believed to result from an alternative splice event that, on the basis of conflicting data obtained in humans, has been considered to be tissue-, developmentally, and/or individual-specific. To decipher the molecular basis of this unusual trait, we isolated a 6.8-kilobase fragment spanning exon 3 from individuals expressing GHRfl. Sequence analysis revealed the existence of two 99% identical retroelements flanking this exon. Unexpectedly, individuals expressing GHRd3 displayed a 2.7-kilobase deletion involving exon 3, which most likely results from an ancestral homologous recombination between the two retroelements. The lineage of these retroelements during primate evolution revealed the species specificity of the GHRd3 allele. These findings led us to propose a model underlying the existence of the sole GHRfl allele in most species. Such a retrovirus-mediated alternative splice mimicry, which clears up several as yet unexplained phenomena (i.e. the above-mentioned expression data, the Mendelian inheritance of GHR expression patterns, and the deletion of nonconsecutive exons in growth hormone resistant patients), represents a novel physiological mechanism accounting for protein diversity between and within species.

Evaluation of parental mitochondrial inheritance in neonates born after intracytoplasmic sperm injection.

Intracytoplasmic sperm injection (ICSI) is now used when severe male-factor infertility has been documented. Since defective mitochondrial functions may result in male hypofertility, it is of prime importance to evaluate the risk of paternal transmission of an mtDNA defect to neonates. DNA samples from the blood of 21 infertile couples and their 27 neonates born after ICSI were studied. The highly polymorphic mtDNA D-loop region was analyzed by four PCR-based approaches. With denaturing gradient gel electrophoresis (DGGE), which allows 2% of a minor mtDNA species to be detected, the 27 newborns had a DGGE pattern identical to that of their mother but different from that of their father. Heteroplasmy documented in several parents and children supported an exclusive maternal inheritance of mtDNA. The parental origin of the children's mtDNA molecules also was studied by more-sensitive assays: restriction-endonuclease analysis (REA) of alpha[32P]-radiolabeled PCR products; paternal-specific PCR assay; and depletion of maternal mtDNA, followed by REA. We did not detect paternal mtDNA in nine neonates, with a sensitivity level of 0.01% in five children, 0.1% in two children, and 1% in two children. The estimated ratio of sperm-to-oocyte mtDNA molecules in humans is 0.1%-1.5%. Thus, we conclude that, in these families, the ICSI procedure performed with mature spermatozoa did not alter the uniparental pattern of inheritance of mtDNA.

Human Prop-1: cloning, mapping, genomic structure. Mutations in familial combined pituitary hormone deficiency.

Prop-1 is a newly isolated pituitary-specific paired-like homeodomain transcription factor whose cDNA sequence is well known in mouse. To study its involvement in human combined pituitary hormone deficiency (CPHD), we have isolated the human cDNA ortholog and determined the exon/intron organization and chromosomal localization of the human gene. A Prop-1 defect was characterized in three CPHD families. One missense mutation (R73C) involves a residue conserved in 95% of the more than 400 homeodomain proteins so far identified; in vitro splicing assays demonstrated the functional importance of the second defect, whereas the remaining mutation is a frameshift. Given the disease phenotype documented in the patients, these data, which will facilitate molecular investigations in other patients, demonstrate the crucial role of Prop-1 in the proper development of somatotrophs, lactotrophs, thyreotrophs and gonadotrophs.

Changes in serum IGF-I and IGFBP-3 concentrations during the IGF-I generation test performed prospectively in children with short stature.

OBJECTIVE: Genotype and phenotype heterogeneity in patients with GH insensitivity syndrome suggests that partial defects exist in the GH receptor. Children with partial GH resistance would be expected to have short stature, elevated GH levels and relatively low levels of IGF-I and IGFBP-3. Provocation tests of the GH-IGF-I axis may help to identify such children. The IGF-I generation test in particular may demonstrate impaired secretion of IGF-I and IGFBP-3. This prospective study assesses the usefulness of the IGF-I generation test in the identification of short children with possible GH insensitivity. DESIGN: Prepubertal children referred for assessment of short stature underwent a standard GH provocation test followed by an IGF-I generation test. SUBJECTS: Thirty-seven prepubertal children (14 girls, 23 boys) with short stature (height < 2nd centile UK standards 1990) aged 4.5-12.6 years were investigated prospectively. METHODS: Assessment included history, physical examination, auxological observations (height, weight, bone age). GH provocation tests (glucagon 15 micrograms/kg i.m. or insulin 0.15 U/kg/i.v.) was followed by an IGF-I generation test (hGH 0.1 iu/kg/s.c. daily for 4 days). MEASUREMENTS: GH was assayed during the provocation test. IGF-I and IGFBP-3 were measured at 0900 h on day 0 and 4 of the IGF-I generation test. GH and IGF-I were measured by radioimmunoassay, IGFBP-3 by IRMA and basal GHBP by HPLC. STATISTICAL ANALYSIS: Height SDS was calculated according to the UK Height Standards 1990. The absolute and percentage changes of IGF-I and IGFBP-3 during the IGF-I generation test were calculated. RESULTS: The 37 children were divided into three groups according to the peak GH level (mean +/- SEM) during the provocation test: Group 1 (peak GH < 20 mU/l) n = 11, five girls, six boys age 7.1 +/- 0.7 years, height SDS -2.5 +/- 0.1, peak GH 14.5 +/- 1.6 mU/l, IGF-I 92.0 +/- 10.4 micrograms/l, IGFBP-3 2.6 +/- 0.4 mg/l. Group 2 (peak GH 20-40 mU/l) n = 12, six girls, six boys age 8.6 +/- 0.7 years, height SDS -2.6 +/- 0.1, peak GH 28.4 +/- 1.6 mU/l, IGF-I 121-5 +/- 13.4 micrograms/l, IGFBP-3 2.9 +/- 0.2 mg/l. Group 3 (peak GH > 40 mU/l) n = 14, three girls, 11 boys, aged 8.5 +/- 0.6 years, height SDS -2.3 +/- 0.1, peak GH 60.7 +/- 4.1 mU/l, IGF-I 112.4 +/- 10.9 micrograms/l, IGFBP-3 3.1 +/- 0.3 mg/l. There were no significant differences in the absolute increases of IGF-I or IGFBP-3 (mean +/- SEM) during the IGF-I generation test, IGF-I; Group 1, 48.8 +/- 9.5 micrograms/l, Group 2, 42.7 +/- 4.8 micrograms/l. Group 3, 45.5 +/- 5.1 micrograms/l, IGFBP-3; Group 1, 1.1 +/- 1.2 mg/l. Group 2, 1.2 +/- 0.2 mg/l, Group 3, 0.85 +/- 0.1 mg/l. There were no significant differences in the percentage increases (mean +/- SEM) of IGF-I; Group 1, 55 +/- 9%, Group 2, 35 +/- 5%, Group 3, 42 +/- 8%, or IGFBP-3; Group 1, 64 +/- 17%, Group 2, 44 +/- 8%, Group 3.32 +/- 6%. GHBP values were normal in all three groups. In Group 3 (peak GH > 40 mU/l) four individual patients had either low basal IGF-I levels (n = 2) (< 5th centile of normal range for age) or low basal IGFBP-3 levels (n = 1) (< 5th centile of normal range for age) or low IGF-I responses in the IGF-I generation test (2 x CV of IGF-I assay) (n = 1). No single subject had all the characteristics of GH insensitivity syndrome. CONCLUSION: The responses during an IGF-I generation test did not identify a clear group of children with GH insensitivity. Individual patients had low basal IGF-I or IGFBP-3 values and a poor response in the generation test, features which, in the presence of high GH levels on provocation, are consistent with partial GH insensitivity.

Evolutionary divergence of the truncated growth hormone receptor isoform in its ability to generate a soluble growth hormone binding protein.

The soluble growth hormone binding protein (GHBP), which is encoded by the GH receptor (GHR) gene, is generated by several mechanisms. In rabbits (rb) and humans (h), it is derived by proteolytic cleavage of the full-length membrane-bound receptor molecules (GHR-fl), whereas in rats (r) and mice, it results from an alternative splice excluding the transmembrane domain. Furthermore, in all these species, alternative splicing in the cytoplasmic domain results in a truncated isoform (GHR-tr), that, in humans, produces large amounts of GHBP through proteolysis. To further characterize the species specificity of the mechanism underlying GHBP generation, rbGHR-tr and rGHR-tr expressed in COS-7 cells were assayed for their ability to produce a GHBP in comparison with the corresponding full-length receptors. Large amounts of GHBP were secreted by cells expressing the rabbit constructs, the rbGHR-tr isoform being more efficient in GHBP generation than rbGHR-fl. In contrast, no GHBP was detected from cells expressing rGHR-tr, the cytoplasmic deletion having no effect on GHBP release from membrane receptors. These data further demonstrate evolutionary divergence in the mechanism by which GHBP is generated and provide new clues to decipher the molecular process underlying the cleavage step.

A new c-kit mutation in a case of aggressive mast cell disease.

Systemic mast cell disease (SMCD) is a disorder characterized by a mast cell proliferation in various tissues. Mast cells express the c-kit proto-oncogene. A few cases of c-kit mutations have been described in SMCD. We report an aggressive SMCD in a patient who presented with a bone marrow infiltration by abnormal mast cells. Molecular studies of mast cell DNA and RNA revealed a new c-kit heterozygous mutation (Asp820Gly). This mutation leads to a drastic amino-acid change and is located close to the highly oncogenic Asp816Val. These findings suggest that the Asp820Gly has a potential role in c-kit activation.

Nine novel growth hormone receptor gene mutations in patients with Laron syndrome.

The GH receptor (GHR) is a member of the cytokine receptor superfamily; GH binding protein is the solubilized extracellular domain of the GHR. Abnormalities in the GHR produce an autosomal recessive form of GH resistance, the Laron syndrome, characterized by growth failure and the clinical appearance of severe GH deficiency despite elevated circulating GH levels. In 13 unrelated patients with undetectable levels of GH binding protein, we characterized nine novel mutations in the GHR gene. These molecular defects comprise three nonsense mutations (Q65X, W80X, and W157X), one frameshift (36delC), two splice defects (G-->A at 70 + 1, C-->T at 723), and three missense mutations (C38S, S40L, and W50R) located in the extracellular domain of the receptor, and thus would be expected to interfere with GH binding activity. These results further confirm the broad heterogeneity of mutations underlying this rare GH resistance syndrome.

Alternatively spliced forms in the cytoplasmic domain of the human growth hormone (GH) receptor regulate its ability to generate a soluble GH-binding protein.

The mechanism underlying the generation of soluble growth hormone binding protein (GHBP) probably differs among species. In rats and mice, it involves an alternatively spliced mRNA, whereas in rabbits, it involves limited proteolysis of the membrane-bound growth hormone receptor (GHR). In humans, this latter mechanism is favored, as no transcript coding for a soluble GHR has been detected so far. To test this hypothesis, we analyzed COS-7 cells transiently expressing the full-length human (h) GHR and observed specific GH-binding activity in the cell supernatants. Concomitantly, an alternatively spliced form in the cytoplasmic domain of GHR, hGHR-tr, was isolated from several human tissues. hGHR-tr is identical in sequence to hGHR, except for a 26-bp deletion leading to a stop codon at position 280, thereby truncating 97.5% of the intracellular domain of the receptor protein. When compared with hGHR, hGHR-tr showed a significantly increased capacity to generate a soluble GHBP. Interestingly, this alternative transcript is also expressed in liver from rabbits, mice, and rats, suggesting that, in these four species, proteolysis of the corresponding truncated transmembrane GHR is a common mechanism leading to GHBP generation. These findings support the hypothesis that GHBP may at least partly result from alternative splicing of the region encoding the intracellular domain and that the absence of a cytoplasmic domain may be involved in increased release of GHBP.

Molecular basis of inherited growth hormone resistance in childhood.

The growth hormone receptor (GHR), a member of the cytokine receptor superfamily that gives rise to a soluble and circulating counterpart (GHBP), is the main target of Laron syndrome (LS), a severe autosomal recessive dwarfism characterized by complete GH insensitivity. Genetic and mutation analyses have attested to the high molecular heterogeneity of this syndrome, and, to date, more than 30 different GHR mutations including deletion, frameshift, nonsense, missense and splicing defects have been described. However, among them, missense mutations are of particular interest in potentially providing critical information on the structure-function relationship of the GHR and related molecules. The study of the recently described forms of atypical LS is now very promising. These patients display detectable plasma GH binding activity associated with complete or partial GH insensitivity. Molecular analysis of such a phenotype with positive GHBP and complete GH insensitivity has revealed the existence of a missense mutation abolishing receptor homodimerization, thereby providing in vivo evidence for the critical role of the dimerization process in the growth-promoting action of GH. Similarly, mutations in the cytoplasmic region, which are expected to be associated with normal GH binding activity, should contribute to the identification of other functionally important domains. Partial GH insensitivity syndromes may theorically encompass a wide range of distinct phenotypes with variable degrees of GH resistance. Missense GHR mutations and a quantitative GHR mRNA defect have been identified in some cases belonging to this heterogeneous group. Interestingly, exclusion of linkage between the Laron phenotype and the GHR locus was demonstrated in one affected family. This latter situation may indicate the existence of other genes controlling GHR expression or required at different steps of the signal transduction pathway. In this regard, the availability of a possible animal model for LS should offer new prospects in the identification of GH-inducible genes.

Isolated growth hormone deficiency type IA associated with a 45-kilobase gene deletion within the human growth hormone gene cluster in an Italian family.

An Italian family with three children presenting with isolated growth hormone (GH) deficiency type IA is described. Restriction endonuclease analysis revealed that the cause of hGH deficiency was a 45-kb gene deletion within the hGH-chorionic somatomammotropin (CS) gene cluster, encompassing the GH-1, CS-L, CS-A, and GH-2 genes. DNA sequence analysis and polymerase chain reaction amplification between two sequences located on each side of the deletion breakpoint accurately identified the deletion breakpoints and indicated that the regulatory sequences located upstream from the TATA box of the mutant CS-B belong to the GH-2 gene. Two of the affected children developed high-titer anti-hGH antibodies after recombinant hGH treatment with secondary growth arrest, whereas the third one maintained normal growth in the presence of very low-titer antibodies. This is the first report of a large deletional mutation within the hGH-CS gene cluster accompanied by phenotypic heterogeneity in terms of growth response and antibody formation in the different patients.

An exon-skipping mutation in the btk gene of a patient with X-linked agammaglobulinemia and isolated growth hormone deficiency.

X-linked agammaglobulinemia (XLA) is an inherited immunodeficiency disease associated with a block in differentiation from pre-B to B cells. The XLA gene encodes a 659 amino acids cytoplasmic protein tyrosine kinase named btk (Bruton's tyrosine kinase). The few btk gene alterations so far reported in XLA patients are heterogenous and distributed in all domains of the btk protein. They appear to be responsible for a range of B cell immunodeficiency disorders of variable severity. Rare families in which XLA is inherited together with isolated growth hormone deficiency (IGHD) have been reported. Genetic analysis has shown that this disease association maps to the same region of the X chromosome as XLA, but whether the two phenotypes are caused by a common or different developmental or biochemical mechanism is unknown. We have analyzed the btk gene of a patient with XLA and IGHD. RT-PCR analysis of btk transcripts, sequencing data obtained from cDNA and genomic DNA and in vitro splicing assays showed that an intronic point mutation (1882 + 5G-->A) is responsible for skipping of an exon located in the tyrosine kinase domain. This exon-skipping event results in a frameshift leading to a premature stop codon 14 amino acids downstream, and in the loss of the last 61 residues of the carboxy-terminal end of the protein. Although we studied a sporadic case, the results suggest that an alteration of the btk gene might cause this unusual phenotype.

The insulin-like growth factor I generation test in the investigation of short stature.

Genotypic and phenotypic heterogeneity in patients with growth hormone (GH) insensitivity syndrome suggests that partial defects exist in the GH receptor. The insulin-like growth factor I (IGF-I) generation test was assessed as a means of identifying partial GH receptor defects in a heterogeneous group of 22 prepubertal children with short stature. In a subgroup of nine patients with peak GH levels of 63.7 +/- 3.7 mU/l during a glucagon tolerance test, the response to the IGF-I generation test was no different from that for the group as a whole (peak GH, 43.3 +/- 4.5 mU/l), despite the fact that this subgroup exhibited a negative relationship between height SDS and peak GH and a positive relationship between height SDS and IGF binding protein-3. This preliminary study therefore suggests that the IGF-I generation test in its present form will not be useful as a primary screening test for partial GH insensitivity. Despite this, the IGF-I generation test has been extremely useful in the confirmation of the diagnosis of GHIS and may therefore also prove useful in the confirmation of partial defects in the GH receptor. A subgroup of short children with peak GH levels above 40 mU/l had some characteristics of partial GH receptor deficiency. These children, to whom GH therapy would not normally be given, may respond better to recombinant human IGF-I.

A single amino acid substitution in the exoplasmic domain of the human growth hormone (GH) receptor confers familial GH resistance (Laron syndrome) with positive GH-binding activity by abolishing receptor homodimerization.

Growth hormone (GH) elicits a variety of biological activities mainly mediated by the GH receptor (GHR), a transmembrane protein that, based on in vitro studies, seemed to function as a homodimer. To test this hypothesis directly, we investigated patients displaying the classic features of Laron syndrome (familial GH resistance characterized by severe dwarfism and metabolic dysfunction), except for the presence of normal binding activity of the plasma GH-binding protein, a molecule that derives from the exoplasmic-coding domain of the GHR gene. In two unrelated families, the same GHR mutation was identified, resulting in the substitution of a highly conserved aspartate residue by histidine at position 152 (D152H) of the exoplasmic domain, within the postulated interface sequence involved in homodimerization. The recombinant mutated receptor protein was correctly expressed at the plasma membrane. It displayed subnormal GH-binding activity, a finding in agreement with the X-ray crystal structure data inferring this aspartate residue outside the GH-binding domain. However, mAb-based studies suggested the critical role of aspartate 152 in the proper folding of the interface area. We show that a recombinant soluble form of the mutant receptor is unable to dimerize, the D152H substitution also preventing the formation of heterodimers of wild-type and mutant molecules. These results provide in vivo evidence that monomeric receptors are inactive and that receptor dimerization is involved in the primary signalling of the GH-associated growth-promoting and metabolic actions.

A homozygous insertion-deletion in the type VII collagen gene (COL7A1) in Hallopeau-Siemens dystrophic epidermolysis bullosa.

The Hallopeau-Siemens type of recessive dystrophic epidermolysis bullosa (HS-RDEB) is a life-threatening autosomal disease characterized by loss of dermal-epidermal adherence with abnormal anchoring fibrils (AF). We recently linked HS-RDEB to the type VII collagen gene (COL7A1) which encodes the major component of AF. We describe a patient who is homozygous for an insertion-deletion in the FN-4A domain of the COL7A1 gene. This defect causes a frameshift mutation which leads to a premature stop codon in the FN-5A domain, resulting in a marked diminution in mutated mRNA levels, with no detectable type VII collagen polypeptide in the patient. Our data suggest strongly that this null allele prevents normal anchoring fibril formation in homozygotes and is the underlying cause of HS-RDEB in this patient.