[Clinical, biological and genetic heterogeneity of the inborn errors of pulmonary surfactant metabolism: SP-B deficiency and alveolar proteinosis]. / Hétérogénéité clinico-biologique et génétique des anomalies innées du métabolisme du surfactant pulmonaire: déficit en SP-B et protéinose alvéolaire.

Pulmonary surfactant is a multimolecular complex located at the air-water interface within the alveolus and to which a bulk of functions has been assigned, physical (surface-active properties) as well as immune or depurant. This complex consists of a surface active lipid layer (mainly phospholipids), and of an aqueous subphase. From discrete surfactant sub-fractions, one can isolate very hydrophobic proteins SP-B and SP-C as well as the collectins SP-A and SP-D, which were shown to have structural, metabolic, or defensive properties. Inborn or acquired abnormalities of surfactant, qualitative or quantitative in nature, account for a number human diseases. Beside hyaline membrane disease of the preterm neonate, a cluster of hereditary or acquired lung diseases have been characterized by the storage of periodic acid Schiff-positive material filling the alveoli. From this heterogeneous nosologic bulk, at least two discrete entities presently seem to emerge: 1) SP-B deficiency, in which an essentially proteinaceous material is stored within the alveoli, and which is a bona fide autosomal recessive Mendelian entity linked to the SFTPB gene (MIM 1786640), generally entailing neonatal respiratory distress with rapid fatal outcome, although partial or transient deficiencies have also been observed; 2) alveolar proteinosis, characterized by the storage of a mixed, protein and lipid material, and which constitutes a relatively heterogeneous clinical biological syndrome, with regards to age at onset (from the neonate through to adulthood) as well as the severity of associated signs. Murine models with a targeted mutation of the gene encoding GM-CSF (Csfgm) or the beta subunit of its receptor (Il3rbl) support the hypothesis of an abnormality of surfactant turnover in which the alveolar macrophage would be a key player. Beside SP-B deficiency, in which a near-consensus diagnostic chart can be designed, the ascertainment of other abnormalities of surfactant metabolism is not straightforward. The disentanglement of this disease cluster is however essential, with aim to propose differentiated therapeutic procedure : repeated bronchoalveolar lavages, GM-CSF replacement, bone marrow grafting or lung transplantation.

[Constitutional deficiency of pulmonary surfactant protein B: clinical presentation, histologic and molecular diagnosis]. / Déficit constitutionnel en protéine B du surfactant pulmonaire: présentation clinique, diagnostic histologique et moléculaire.

We report a female full-term infant with fatal respiratory failure of early onset due to inherited SP-B deficiency. Lung biopsy was performed at 18 days after birth, with histopathological characterization indicating congenital alveolar proteinosis. Immunohistochemical studies of lung tissue revealed the absence of SP-B and the presence of intra-alveolar SP-A normal quantities. Analysis of genomic DNA showed homozygosity for the 121ins2 mutation of the SFTPB gene. The infant died 21 days after birth. Both parents were heterozygotes for the mutation. Chorionic villus sampling was performed at the first trimester of the following pregnancy. Restriction analysis of amplified fetal DNA, studies of microsatellite segregation and direct sequencing led to the diagnosis of homozygosity for the parental wild-type allele. The diagnosis of congenital SP-B deficiency should be suspected whenever an early and acute respiratory failure in a term or near-term infant does not resolve after five days of age: diagnostic confirmation can be easily and rapidly obtained with the analysis of genomic DNA and immunohistochemical characterization of lung tissue.

Anti-GM-CSF antibodies in paediatric pulmonary alveolar proteinosis.

BACKGROUND: Auto-antibodies against granulocyte-macrophage colony stimulating factor (GM-CSF) may be central to the pathogenesis of adult sporadic pulmonary alveolar proteinosis (PAP). The role of anti-GM-CSF auto-antibodies in paediatric forms of PAP is as yet unclear. METHODS: Anti-GM-CSF auto-antibodies were determined with the help of an antigen capture assay using serum and/or bronchoalveolar lavage (BAL) fluid from 27 patients with PAP (nine adults, 15 children, three neonates) and from 185 children with different diseases as disease controls (various pulmonary conditions and patients with malignancies). RESULTS: Anti-GM-CSF auto-antibodies were detected in the serum of five of seven adult PAP patients. They were not found in the serum of any of the children or neonates with PAP nor in any of the disease control patients. Raised anti-GM-CSF titres were found in BAL fluid from three of four adult patients with PAP. Anti-GM-CSF auto-antibodies were detected in BAL fluid of only one of the 15 children (age at diagnosis 11 years, age at BAL 24 years) and in none of the neonates with PAP, nor in any of the disease control patients. CONCLUSIONS: The presence of anti-GM-CSF auto-antibodies seems to define an autoimmune disease underlying most of the adult sporadic type of PAP, but age at diagnosis may cause an overlap with children in some rare instances. In most of the children and all of the neonates the anti-GM-CSF titres were not significantly increased, indicating that alternative explanations are needed for the pathogenesis of the disease in these patients.

FOXC2 truncating mutation in distichiasis, lymphedema, and cleft palate.

We report a family showing autosomal-dominant segregation of upper- and lower-eyelid distichiasis (double row of eyelashes) in seven affected relatives over three generations, in addition to below-knee lymphedema of pubertal onset (lymphoedema proecox) in three. Two children had cleft palate in addition to distichiasis, but without the previously reported association with the Pierre-Robin sequence. Other ophthalmologic anomalies included divergent strabismus and early-onset myopia. This family was found to be completely linked to markers mapped to 16q24.3 and thereby proposed to be allelic to the distichiasis-lymphedema syndrome (DL, MIM 153400), although pterygium colli, congenital heart disease, or facial dysmorphism were not features found here. As FOXC2/FKLH14 mutations were found to underlie DL and diverse hereditary lymphedema conditions, this gene was examined by sequence analysis. An out-of-frame deletion (914-921del) was identified and found to segregate with the disease, further highlighting the phenotypic heterogeneity of lymphedema conditions linked to FOXC2 truncating mutations. Whether such heterogeneity is related to genotype-phenotype correlation, a hypothesis not primarily supported by the apparent loss-of-function mechanism of the mutations, or governed by modifying genes, remains to be determined.

Identification of a novel Ca2+-stimulated S6-kinase in rat liver.

Extracellular calcium addition transiently stimulated two S6 peptide kinase activities in isolated rat hepatocytes. Mono Q chromatography revealed that the activities eluting at 0.15 M NaCl and 0.18 M NaCl were stimulated 4-fold and 2-fold, respectively. The kinase stimulated by calcium was a 40000-Mr S6 peptide kinase, as demonstrated by partial purification from whole liver. The protein kinase did not crossreact with antibodies directed against the N- or C-terminal part of p70 ribosomal S6 kinase (p70(S6K)) and the C-terminal part of p90 ribosomal S6 kinase (p90(rsk)). Following digestion of 40000-Mr S6 peptide kinase with trypsin, six peptides were sequenced. There was no similarity with the sequences of p70(S6K) and p90(rsk). Moreover, the obtained sequences could not be identified in the SwissProt or EMBL-genebank databases, suggesting that 40000-Mr S6 peptide kinase probably represents a novel protein kinase.

Clinical biological and genetic heterogeneity of the inborn errors of pulmonary surfactant metabolism.

Pulmonary surfactant is a multimolecular complex located at the air-water interface within the alveolus to which a range of physical (surface-active properties) and immune functions has been assigned. This complex consists of a surface-active lipid layer (consisting mainly of phospholipids), and of an aqueous subphase. From discrete surfactant sub-fractions one can isolate strongly hydrophobic surfactant proteins B (SP-B) and C (SP-C) as well as collectins SP-A and SP-D, which were shown to have specific structural, metabolic, or immune properties. Inborn or acquired abnormalities of the surfactant, qualitative or quantitative in nature, account for a number of human diseases. Beside hyaline membrane disease of the preterm neonate, a cluster of hereditary or acquired lung diseases has been characterized by periodic acid-Schiff-positive material filling the alveoli. From this heterogeneous nosologic group, at least two discrete entities presently emerge. The first is the SP-B deficiency, in which an essentially proteinaceous material is stored within the alveoli, and which represents an autosomal recessive Mendelian entity linked to the SFTPB gene (MIM 1786640). The disease usually generally entails neonatal respiratory distress with rapid fatal outcome, although partial or transient deficiencies have also been observed. The second is alveolar proteinosis, characterized by the storage of a mixed protein and lipid material, which constitutes a relatively heterogeneous clinical and biological syndrome, especially with regard to age at onset (from the neonate through to adulthood) as well as the severity of associated signs. Murine models, with a targeted mutation of the gene encoding granulocyte macrophage colony-stimulating factor (GM-CSF) (Csfgm) or the beta subunit of its receptor (II3rb1) support the hypothesis of an abnormality of surfactant turnover in which the alveolar macrophage is a key player. Apart from SP-B deficiency, in which a near-consensus diagnostic chart can be designed, the ascertainment of other abnormalities of surfactant metabolism is not straightforward. The disentanglement of this disease cluster is however essential to propose specific therapeutic procedures: repeated broncho-alveolar lavages, GM-CSF replacement, bone marrow grafting or lung transplantation.

Interstitial lung disease in a baby with a de novo mutation in the SFTPC gene.

Mutations in the surfactant protein C gene (SFTPC) were recently reported in patients with interstitial lung disease. In a 13-month-old infant with severe respiratory insufficiency, a lung biopsy elicited combined histological patterns of nonspecific interstitial pneumonia and pulmonary alveolar proteinosis. Immunohistochemical and biochemical analyses showed an intra-alveolar accumulation of surfactant protein (SP)-A, precursors of SP-B, mature SP-B, aberrantly processed proSP-C, as well as mono- and dimeric SP-C. Sequencing of genomic DNA detected a de novo heterozygous missense mutation of the SFTPC gene (g.1286T>C) resulting in a substitution of threonine for isoleucine (173T) in the C-terminal propeptide. At the ultrastructural level, abnormal transport vesicles were detected in type-II pneumocytes. Fusion proteins, consisting of enhanced green fluorescent protein and wild-type or mutant proSP-C, were used to evaluate protein trafficking in vitro. In contrast to wild-type proSP-C, mutant proSP-C was routed to early endosomes when transfected into A549 epithelial cells. In contrast to previously reported mutations, the 173T represents a new class of surfactant protein C gene mutations, which is marked by a distinct trafficking, processing, palmitoylation, and secretion of the mutant and wild-type surfactant protein C. This report heralds the emerging diversity of phenotypes associated with the expression of mutant surfactant C proteins.

Improved fluorescent PCR-based assay for sizing CGG repeats at the FRAXA locus.

Fragile X syndrome is the most frequent heritable genetic disease involving mental retardation and is usually caused by an expanded CGG repeat in the first exon of the FMR1 gene. Therefore, searching for CGG expansion at the FRAXA locus among the mentally retarded has become a routine investigation in neuro-paediatric practice. Consequently, we have developed a fluorescent PCR-based assay for sizing repeats as an alternative to laborious and time-consuming Southern blot. The procedure utilises a reverse fluorescent labelled primer, and the Expand Long Template PCR system (Roche) with addition of dimethylsulfoxide and 7-deaza-dGTP It allows precise determination of the CGG repeat number in males and females for alleles from normal to premutation size range and detection of full mutations in males. We believe that this PCR protocol, allowing a high sample throughput, is useful for first-line screening among mentally retarded males, possibly complemented by Southern blot analysis to assess the methylation status of large mutated alleles.

Linkage analysis of 5 novel van der Woude syndrome kindreds to 1q32-q41 markers further supports locus homogeneity of the disease trait.

van der Woude syndrome (vWS, MIM 119300) is a rare autosomal dominant clefting condition with cardinal features of mucous cysts (lower-lip pits) and clefts to the lip and/or palate. The vWS gene has been assigned to a locus in 1q32-q41 by linkage analysis and physical mapping. We have investigated 5 novel vWS families through probands attended for cleft lip and/or palate repair at the Department of Maxillofacial Surgery of Hôpital Trousseau, Paris, in order to tentatively refine the genetic map of the vWS region in 1q32-q41 and possibly identify unlinked pedigrees. Linkage analysis was carried out to 6 microsatellite markers (D1S249, D1S425, D1S491, D1S205, D1S414, D1S425), yielding a maximum cumulative LOD score of Z = 3.27 at theta = 0.00 for D1S245. The innermost four markers were found to be tightly linked to one another, with no evidence for recombination. Our results support linkage of vWS within a region of tightly linked markers and do not favour locus heterogeneity of the disease trait.

Compound SFTPB 1549C-->GAA (121ins2) and 457delC heterozygosity in severe congenital lung disease and surfactant protein B (SP-B) deficiency.

Several human respiratory disorders have been linked to an abnormality of pulmonary surfactant synthesis or turnover. Among those conditions, hereditary deficiency in the hydrophobic surfactant protein B (SP-B) has been recognized as a rare cause of respiratory failure in term newborn infants. Homozygosity for a common mutation (1549C-->GAA, or 121ins2) of the SP-B-encoding gene (SFTPB) results in rapidly fatal respiratory failure, with complete absence of the mRNA and protein observed in lung fluid or biopsy specimens. Hereditary SP-B deficiency is also associated with aberrant processing of proSP-C and deficiency of the active SP-C peptide. In the present study, we characterized the SFTPB gene in an infant with severe unexplained respiratory distress and identified a paternally derived 1549C-->GAA lesion, as well as a hitherto unreported mutation (457delC) inherited from the mother. Analysis of bronchoalveolar lavage fluid demonstrated the complete absence of SP-B. However, unlike previous infants with hereditary SP-B deficiency, proSP-C was processed to the active SP-C peptide, suggesting that the defect in SP-B, rather than SP-C, caused the respiratory distress in this infant. The present findings demonstrate the importance of SFTPB in pulmonary function and support the need for further genotype-phenotype correlations in patients with SP-B deficiency.