CFTR mutations and IVS8-5T variant in newborns with hypertrypsinaemia and normal sweat test.

Neonates positive for immunoreactive trypsinogen assay (IRT) and negative for sweat test have formerly been found to carry the major cystic fibrosis (CF) mutation, delta F508, much more frequently than the general population. Among the 716 IRT positive newborns detected by a three tier (IRT, mutation analysis plus meconium lactase assay, sweat test) CF screening programme in north eastern Italy during the period January 1993 to March 1996, we found 45 carriers, a number significantly higher than the expected 17 (p < 0.001). We speculated that some of these heterozygotes could actually be affected by a very mild form of CF, and carry on the other chromosome an undetected CFTR mutation or a DNA variant, such as the 5-thymidine allele in intron 8 of the CFTR gene (IVS8-5T). This hypothesis was tested in four samples; group A (the 45 carriers mentioned above), group B (51 non-carrier, IRT positive neonates), group C (50 IRT negative neonates), and group D (90 CF adult female carriers). Chromosomes with IVS8-5T were seven (7.78%) in group A, seven (6.86%) in group B, five (5%) in group C, and four in group D (2.22%). The 5T prevalence in group A was significantly higher (p < 0.05) compared to group D; similarly, a higher (p < 0.05) 5T frequency in group A compared to group C was detected by considering the chromosomes free from CFTR mutations. This study is consistent with previous papers in finding among neonates with high trypsin levels a CF carrier frequency significantly higher than that expected. It is also suggested that in at least some babies raised trypsin levels at birth could be a phenotypic expression of compound heterozygosity for a major CF mutation plus IVS8-5T.     

Interaction between a novel TGFB1 haplotype and CFTR genotype is associated with improved lung function in cystic fibrosis

Cystic fibrosis (CF), the most common lethal single gene disorder in Caucasians, is due to mutations in the CFTR gene. Twin and sibling analysis indicates that modifier genes, rather than allelic variation in CFTR, are responsible for most of the variability in severity of lung disease, the major cause of mortality in CF patients. We used a family-based approach to test for association between lung function and two functional SNPs (rs1800469, '-509' and rs1982073, 'codon 10') in the 5' region of transforming growth factor-beta1 (TGFB1), a putative CF modifier gene. Quantitative transmission disequilibrium testing of 472 CF patient-parent-parent trios revealed that both TGFB1 SNPs showed significant transmission distortion when patients were stratified by CFTR genotype. Although lung function and nutritional status are correlated in CF patients, there was no evidence of association between the TGFB1 SNPs and variation in nutritional status. Additional tagging SNPs (rs8179181, rs2278422, rs8110090, rs4803455 and rs1982072) that capture most of the diversity in TGFB1 were also typed but none showed association with variation in lung function. However, a haplotype composed of the -509 C and codon 10 T alleles along with the C allele of the 3' SNP rs8179181 was highly associated with increased lung function in patients grouped by CFTR genotype. These results demonstrate that TGFB1 is a modifier of CF lung disease and reveal a previously unrecognized beneficial effect of TGFB1 variants upon the pulmonary phenotype.     

The tRNA(Gln) 4336 mitochondrial DNA variant is not a high penetrance mutation which predisposes to dementia before the age of 75 years.

The genetic factors that predispose to Alzheimer's disease (AD) are heterogeneous. Two recent reports have suggested that a mitochondrial DNA mutation within the tRNAGln gene, located at position 4336, may be a risk factor for AD, as it was found in 10/256 (3.9%) cases with AD confirmed by necropsy. Although low prevalences of this mutation were detected in non-demented subjects in both of these studies, the controls were not carefully matched with the AD cases. We have investigated the frequency of this mutation in two community based elderly cohorts in Cambridgeshire, who have participated in longitudinal studies of cognitive function. The 4336 mitochondrial mutation was detected in 8/ 443 people examined. These people were found to be non-demented at ages 74, 81, 84, 86, 89, 90, 91, and 102 years, in contrast to the previously described cases whose onset of dementia occurred between 60 and 76 years (mean 68). Accordingly, we believe that this mitochondrial variant is not a high penetrance mutation which predisposes to dementia before the age of 76 years.     

CRB1 has a cytoplasmic domain that is functionally conserved between human and Drosophila.

Mutations in the human Crumbs homologue 1 (CRB1) gene cause severe retinal dystrophies, ranging from retinitis pigmentosa to Leber congenital amaurosis. The CRB1 gene is expressed specifically in human retina and brain and encodes a protein homologous to the Drosophila Crumbs protein. In crumbs mutant embryos apico-basal polarity of epithelial cells is lost, leading to widespread epidermal cell death. The small cytoplasmic domain of Crumbs organizes an intracellular protein scaffold that defines the assembly of a continuous zonula adherens. The crumbs mutant phenotype can be partially rescued by expression of just the membrane-bound cytoplasmic domain, and overexpression of this domain in a wild-type background results in a multilayered epidermis. A striking difference between CRB1 and Crumbs was that the latter contains a transmembrane region and a 37 amino acid cytoplasmic domain. Here we describe an alternative splice variant of human CRB1 that encodes a cytoplasmic domain 72% similar to that of Drosophila Crumbs. Two intracellular subdomains that are necessary for function in Drosophila are absolutely conserved. Rescuing and overexpression studies in Drosophila show that the cytoplasmic domains are functionally related between these distant species. This suggests that CRB1 organizes an intracellular protein scaffold in the human retina. Human homologues of proteins binding to Crumbs may be part of this complex and represent candidate genes for retinal dystrophies.     

The murine T-lymphocyte response to tyrosine-azobenzenearsonate. Characteristics of a low responder haplotype T-cell clone

An I-Ab-restricted, L3T4+ Ly2- T-cell clone, 5R-4F3, specific for ABAtyr was established in culture from a B10.A(5R) mouse. Since b haplotype mice respond weakly to ABAtyr compared to other haplotypes, this is a candidate clone of low responder phenotype. In support of this contention, 5R-4F3 grew very poorly under conditions that supported the vigorous growth of E beta bE alpha k-restricted T-cell clones from the same mouse. The I-A (low responder) and I-E (high responder) restricted T-cell clones also differed in their responses to apc pre-pulsed with antigen, compared to apc with antigen present continuously during culture. The low and high responder clones responded comparably to IL-2. Attempts to elevate the response of C57BL/6 mice to ABAtyr in vivo by injecting them with human recombinant IL-2 and antigen together were only partially successful: C57BL/6 mice treated in this way showed a 3-5-fold increase in their proliferative responses to ABAtyr, which was at best only one quarter of the level of response shown by high responder A/J mice to the same antigen dose.     

Evidence that T colony formation is a property of T mu (helper) lymphocytes.

The T-colony-forming capacity of different T lymphocyte subsets was studied in normal peripheral blood. Unfractionated lymphocytes (after 'Lymphoprep' separation) gave rise to a mean of 150 +/- 27 . 7 s.d. T colonies per 1 x 10(5) cells, while purified T lymphocytes by sheep RBC rosetting formed 110 +/- 32 . 2 colonies. Two subpopulations of T lymphocytes were further isolated according to the presence of Fc receptors for IgG (T gamma) or IgM (T mu) by ox RBC rosetting. T gamma cells were found to have a very low or absent T colony-forming capacity (23 +/- 26 . 2), while T mu cells produced normal colony numbers (106 +/- 28 . 4). Co-culture experiments showed that T gamma cells do not inhibit the T colony growth of normal T cells in our system. Our findings indicate that in human peripheral blood not all T lymphocytes are capable of forming T colonies and that this property is confined to the T mu (helper) lymphocyte subset.     

A self-reactive T cell population that is not subject to negative selection.

In male mice expressing a transgenic alpha beta TCR which recognizes a male antigen (HY), T cells which do not express normal levels of CD8 escape thymic deletion and appear in the periphery. These consist of two distinct populations, one which lacks expression of both CD4 and CD8, and one with low levels of CD8. Neither population has anti-HY reactivity, consistent with the known requirement of this TCR for CD8. We now describe the consequences of expression of both the anti-HY TCR transgene and a constitutive CD8.1 transgene on T cells of male mice. Peripheral T cells in these male 'double transgenic' mice express both the anti-HY TCR and normal levels of CD8, and can proliferate to male antigen in vitro. These cells do not express the endogenous allele of CD8 (CD8.2), suggesting that the increase in CD8 levels due to the CD8.1 transgene leads to the deletion of the CD8.2low population. In contrast, the CD8.1 transgene does not lead to the deletion of the CD8.2- population. This implies that, unlike the majority of alpha beta T cells, TCR+CD4-CD8- cells in TCR transgenic mice are not subject to deletion.     

A SImplified method for Segregation Analysis (SISA) to determine penetrance and expression of a genetic variant in a family

A method for SImplified rapid Segregation Analysis (SISA) to assess penetrance and expression of genetic variants in pedigrees of any complexity is presented. For this purpose the probability for recombination between the variant and the gene is zero. An assumption is that the variant of undetermined significance (VUS) is introduced into the family once only. If so, all family members in between two members demonstrated to carry a VUS, are obligate carriers. Probabilities for cosegregation of disease and VUS by chance, penetrance, and expression, may be calculated. SISA return values do not include person identifiers and need no explicit informed consent. There will be no ethical complications in submitting SISA return values to central databases. Values for several families may be combined. Values for a family may be updated by the contributor. SISA is used to consider penetrance whenever sequencing demonstrates a VUS in the known cancer-predisposing genes. Any family structure at hand in a genetic clinic may be used. One may include an extended lineage in a family through demonstrating the same VUS in a distant relative, and thereby identifying all obligate carriers in between. Such extension is a way to escape the selection biases through expanding the families outside the clusters used to select the families.     

Multiple lipomas linked to an RB1 gene mutation in a large pedigree with low penetrance retinoblastoma

Hereditary predisposition to lipomas is observed in familial multiple lipomatosis (OMIM 151900) and benign cervical lipomatosis (OMIM 151800) and can also be associated with mutations in the MEN1 and PTEN genes (OMIM 131100 and 153480, respectively). In addition, a recent report indicates that a few patients with hereditary retinoblastoma also have lipomas. Here we report on an extended family segregating a splice site mutation in the RB1 gene. Almost all adult carriers of this mutation had multiple lipomas while penetrance for retinoblastoma was incomplete. In an unrelated pedigree, which was reported previously, the identical mutation was only associated with low-penetrance retinoblastoma but not lipomas. Our data indicate that lipoma predisposition in hereditary retinoblastoma is not associated with specific RB1 gene mutations but is influenced by modifying factors linked to this gene.     

Evidence that X-linked severe combined immunodeficiency is not a differentiation defect of T lymphocytes.

In order to gain information about the nature of the defect in X-linked severe combined immunodeficiency (XSCID), we investigated gene expression in different lymphoid and haematopoietic cells of female carriers by looking for non-random X chromosome usage. We have shown non-random X chromosome usage in T lymphocyte enriched (E+) fraction in all carriers. E- cells and monocytes showed non-random X chromosome usage in three carriers tested. In the B cell series one carrier showed non-random inactivation in all EBV lines tested (10) and the same X chromosome was shown to be active in all cells. In other carriers there was a preference for use of the normal X chromosome but some B cell lines used the mutant X as well as the normal X. Similar results were found with granulocytes. In two female carriers DNA made directly from whole blood showed a non-random pattern of X chromosome usage. In fibroblast cultures from two female carriers more cells had the normal gene on the active X chromosome than had the defective gene on the active X chromosome. Within families there was heterogeneous expression of the gene. The gene that is defective in XSCID is expressed in all the cell types studied and, therefore, is not a T lymphocyte differentiation gene. The results are consistent with the gene being in a metabolic pathway as in certain autosomal recessive forms of SCID i.e. adenosine deaminase deficiency and purine nucleoside phosphorylase deficiency.     

Heterogeneity of rabbit endogenous pyrogens is not attributable to glycosylated variants of a single polypeptide chain.

Rabbit endogenous pyrogens were of about the same molecular size, but showed considerable heterogeneity of their isoelectric points. We attempted to show that this heterogeneity was attributable to variable glycosylation of a single polypeptide chain. When peritoneal exudate cells were stimulated to make pyrogens in the presence of 2-deoxy-D-glucose, there was a relatively trivial suppression of pyrogen release, and analysis by isoelectric focusing showed parallel inhibition of secretion of all the forms of endogenous pyrogen. When cells were stimulated in the presence of 3H-labeled amino acids and 14C-labeled glucosamine or glucose, the purified pyrogens were labeled with 3H but not with 14C. Macrophage membrane preparations were made which contained glycosyl transferases and could transfer sugar residues from sugar nucleotides to deglycosylated fetuin. These macrophage membrane preparations did not transfer sugars to the pI 7.3 endogenous pyrogen. Treatment of endogenous pyrogens with neuraminidase or with periodate produced no evidence suggesting that the pyrogens were glycosylated. Last, endogenous pyrogens did not bind to any of four lectins with different carbohydrate specificities. This evidence suggests that the heterogeneity of rabbit endogenous pyrogens is not attributable to glycosylation and must have some other cause.     

High prevalence of congenital deafness on Reunion Island is due to a founder variant of LHFPL5

Reunion Island is a French oversea department in the Indian Ocean with 1.6/1000, an estimated prevalence of deafness that is almost double as compared to the mainland France. Twelve children having isolated bilateral prelingual profound deafness along with motor delay attributed to vestibular areflexia were enrolled. Their mean walking age was 19 months. Electroretinography and temporal bone CT-scans were normal in all cases. A novel homozygous frameshift lipoma HMGIC fusion partner-like 5 (LHFPL5) variant c.185delT p.(Phe62Serfs*23) was identified using whole-exome sequencing. It was found in seven families. Four patients from two different families from both Reunion Island and mainland France, were compound heterozygous: c.185delT p.(Phe62Serfs*23) and c.472C > T p.(Arg158Trp). The phenotype observed in our patients completely mimics the hurry-scurry (hscy) murine Tmhs knock-out model. The recurrent occurrence of same LHFPL5 variant in Reunion Island is attributed to common ancestor couple born in 1693.     

A low COMT activity haplotype is associated with recurrent preeclampsia in a Norwegian population cohort (HUNT2)

The etiology of preeclampsia is complex, with susceptibility being attributable to multiple environmental factors and a large genetic component. Although many candidate genes for preeclampsia have been suggested and studied, the specific causative genes still remain to be identified. Catechol-O-methyltransferase (COMT) is an enzyme involved in catecholamine and estrogen degradation and has recently been ascribed a role in development of preeclampsia. In the present study, we have examined the COMT gene by genotyping the functional Val108/158Met polymorphism (rs4680) and an additional single-nucleotide polymorphism, rs6269, predicting COMT activity haplotypes in a large Norwegian case/control cohort (n(cases)= 1135, n(controls)= 2262). A low COMT activity haplotype is associated with recurrent preeclampsia in our cohort. This may support the role of redox-regulated signaling and oxidative stress in preeclampsia pathogenesis as suggested by recent studies in a genetic mouse model. The COMT gene might be a genetic risk factor shared between preeclampsia and cardiovascular diseases.     

A two-marker haplotype in the IRF5 gene is associated with inflammatory bowel disease in a North American cohort

Interferon regulatory factor 5 (IRF5) located on human chromosome 7q32 is associated with many chronic inflammatory disorders. IRF5 is the key regulator of proinflammatory cytokines and type I interferons. We surveyed two cohorts of inflammatory bowel disease (IBD) patients from a North American Consortium. Six single-nucleotide polymorphisms and a 5-base-pair (bp) insertion-deletion (CGGGG indel)polymorphism were investigated. Cytokine secretion was measured in primary lymphocytes after toll-like receptor 9 stimulation. Two-marker haplotypes containing the pairs (rs4728142-CGGGG indel) and (CGGGG indel-rs7808907) were associated with IBD protection (P=2.89 × 10(-6), P=9.32 × 10(-4) (non-Jewish ancestry) and P=4.68 × 10(-8), P=2.50 × 10(-8) (Jewish ancestry)) and IBD risk (P=0.004, P=0.003 (Jewish ancestry), respectively. IRF5 polymorphisms were risk factors for IBD in a single cohort. Interleukin-12-p70 cytokine production was higher (P=0.04) in lymphocytes from controls with two alleles of the 5-bp insertion. IRF5 polymorphisms contribute to the risk profile for Crohn's disease and ulcerative colitis along with ancestry and NOD2 genotypes.