Guidelines for investigating causality of sequence variants in human disease.

The discovery of rare genetic variants is accelerating, and clear guidelines for distinguishing disease-causing sequence variants from the many potentially functional variants present in any human genome are urgently needed. Without rigorous standards we risk an acceleration of false-positive reports of causality, which would impede the translation of genomic research findings into the clinical diagnostic setting and hinder biological understanding of disease. Here we discuss the key challenges of assessing sequence variants in human disease, integrating both gene-level and variant-level support for causality. We propose guidelines for summarizing confidence in variant pathogenicity and highlight several areas that require further resource development.

A role for coding functional variants in HNF4A in type 2 diabetes susceptibility.

AIMS/HYPOTHESIS: Rare mutations in the gene HNF4A, encoding the transcription factor hepatocyte nuclear factor 4α (HNF-4A), account for ~5% of cases of MODY and more frequent variants in this gene may be involved in multifactorial forms of diabetes. Two low-frequency, non-synonymous variants in HNF4A (V255M, minor allele frequency [MAF] ~0.1%; T130I, MAF ~3.0%)-known to influence downstream HNF-4A target gene expression-are of interest, but previous type 2 diabetes association reports were inconclusive. We aimed to evaluate the contribution of these variants to type 2 diabetes susceptibility through large-scale association analysis. METHODS: We genotyped both variants in at least 5,745 cases and 14,756 population controls from the UK and Denmark. We also undertook an expanded association analysis that included previously reported and novel genotype data obtained in Danish, Finnish, Canadian and Swedish samples. A meta-analysis incorporating all published association studies of the T130I variant was subsequently carried out in a maximum sample size of 14,279 cases and 26,835 controls. RESULTS: We found no association between V255M and type 2 diabetes in either the initial (p = 0.28) or the expanded analysis (p = 0.44). However, T130I demonstrated a modest association with type 2 diabetes in the UK and Danish samples (additive per allele OR 1.17 [95% CI 1.08-1.28]; p = 1.5 × 10⁻4), which was strengthened in the meta-analysis (OR 1.20 [95% CI 1.10-1.30]; p = 2.1 × 10⁻5). CONCLUSIONS/INTERPRETATION: Our data are consistent with T130I as a low-frequency variant influencing type 2 diabetes risk, but are not conclusive when judged against stringent standards for genome-wide significance. This study exemplifies the difficulties encountered in association testing of low-frequency variants.

Common variants in HNF-1 alpha and risk of type 2 diabetes.

AIMS/HYPOTHESIS: Mutations in the hepatocyte nuclear factor 1-alpha gene (HNF-1alpha, now known as the transcription factor 1 gene [TCF1]) cause the most common monogenic form of diabetes, MODY3, but it is not known if common variants in HNF-1a are associated with decreased transcriptional activity or phenotypes related to type 2 diabetes, or whether they predict future type 2 diabetes. SUBJECTS AND METHODS: We studied the effect of four common polymorphisms (rs1920792, I27L, A98V and S487N) in and upstream of the HNF-1alpha gene on transcriptional activity in vitro, and their possible association with type 2 diabetes and insulin secretion in vivo. RESULTS: Certain combinations of the I27L and A98V polymorphisms in the HNF-1alpha gene showed decreased transcriptional activity on the target promoters glucose transporter 2 (now known as solute carrier family 2 [facilitated glucose transporter], member 2) and albumin in both HeLa and INS-1 cells. In vivo, these polymorphisms were associated with a modest but significant impairment in insulin secretion in response to oral glucose. Insulin secretion deteriorated over time in individuals carrying the V allele of the A98V polymorphism (n = 2,293; p = 0.003). In a new case-control (n = 1,511 and n = 2,225 respectively) data set, the I27L polymorphism was associated with increased risk of type 2 diabetes, odds ratio (OR) = 1.5 (p = 0.002; multiple logistic regression), particularly in elderly (age > 60 years) and overweight (BMI > 25 kg/m(2)) patients (OR = 2.3, p = 0.002). CONCLUSIONS/INTERPRETATION: This study provides in vitro and in vivo evidence that common variants in the MODY3 gene, HNF-1alpha, influence transcriptional activity and insulin secretion in vivo. These variants are associated with a modestly increased risk of late-onset type 2 diabetes in subsets of elderly overweight individuals.

Altered pathological progression of diaphragm and quadriceps muscle in TNF-deficient, dystrophin-deficient mice.

We have previously demonstrated a role for T cells in Duchenne muscular dystrophy (DMD) using the mdx mouse and have shown that T cell killing of dystrophic muscle can occur through perforin-dependent and perforin-independent mechanisms. In this investigation, we explore the possibility that one perforin-independent mechanism utilized by the T cells is cytokine-based killing, specifically by tumor necrosis factor (TNF). We tested this hypothesis by generating mice that are TNF-deficient and dystrophin-deficient (TNF-/mdx). Body mass and muscle mass of the TNF-/mdx mice were significantly less than TNF+/mdx mice at 8 weeks of age. Creatine kinase levels and overall muscle strength were unchanged. Histopathology measurements showed different results in the diaphragm and quadriceps muscles. These data suggest that removal of TNF in vivo in dystrophic mice has differential effects on diaphragm and quadriceps suggesting that TNF is an unfavorable target for immunotherapy for DMD.

Isolation and characterization of membrane-associated placental proteins.

Membrane-associated proteins (MPs) of the human term placenta (afterbirth) were obtained by extracting the insoluble part of the tissue with solubilizing agents, after the soluble material had been removed by washing with saline. The insoluble residue was subsequently exhaustively extracted first with the nonionic detergent Triton X-100 and then with 6 M urea. In the Triton extract eleven new different membrane-associated antigens could be detected by immunochemical methods; they were designated as MP2A to MP2L. One of these proteins (MP2C) was found to be immunochemically identical with the already described soluble placental protein PP21 [3]. MP1 another antigen detected in the Triton extract later was identified as heart stable alkaline phosphatase. In the urea extract eight different membrane-associated antigens could be identified by immunochemical methods; they were designated as MP3 to MP10. MP3 later was found to be immunochemically identical with laminin. All these membrane-associated proteins have now been isolated to purity and characterized by their physico-chemical properties. Specific antisera to the new proteins were obtained by immunizing animals with the corresponding purified proteins. They were used to detect and quantitate the new proteins in extracts of placentas and other human tissues by immunochemical methods such as gel diffusion tests. The immunocytochemical localization of the new proteins as well as measurement of their concentrations in body fluids by sensitive radioimmunoassays or enzyme immunoassays are presently under investigation.

Isolation and characterization of five new soluble placental tissue proteins (PP22, PP23, PP24, PP25, PP26).

Five new soluble placental tissue proteins (PP22, PP23, PP24, PP25, PP26) were isolated to purity from saline extracts of human term placentas and characterized by their physico-chemical properties. Specific antisera to the new proteins were obtained by immunizing animals with the corresponding purified proteins. They were used to detect and quantitate the new proteins in extracts of placentas and other human tissues by immunochemical methods such as gel diffusion tests. The immunohistochemical localization of the new proteins as well as measurement of their concentrations in body fluids by sensitive radioimmunoassays are presently under investigation.

Immunochemically detected placental proteins and their biological functions.

During the last 20 years a systematic search for proteins occurring in human term placenta (afterbirth) has been performed in our laboratory. As a result more than 30 soluble placental proteins and at least 20 different solubilized antigens apparently derived from the placental membranes have been identified by immunochemical methods in extracts from human term placentas. Most of these proteins have already been isolated to purity and characterized by their physicochemical parameters. Specific antisera to these proteins were obtained by immunizing animals with the corresponding purified proteins. They were used detect and localize these antigens by immunochemical methods in the placenta and in other human tissues. Sensitive immunochemical assays have been developed to exactly quantitate the new proteins in body fluids and to find out the diagnostic significance of measurement of these proteins in pregnant women and in patients with tumors and other diseases. Another aim was to elucidate the biological functions of our immunochemically detected proteins. The results obtained thus far are reported.

Isolation and characterization of a flavin-containing placental protein (PP3).

Placental protein 3 (PP3) has been isolated to purity from saline extracts of human term placentas. PP3 turned out to be a flavin-containing protein and thus appears to be an enzyme. The prosthetic group was identified as flavin-adenine dinucleotide (FAD) which was found to be noncovalently bound to the protein. The physical characterization of PP3 showed that the molecules are composed of two identical subunits having molecular weights of 55000 daltons which are noncovalently linked. Each subunit appears to contain one FAD group. In addition PP3 was found to have an electrophoretic mobility in between the alpha 1 and alpha 2 globulins, an isoelectric point in the range of 5.3-5.7, a sedimentation coefficient of 6.3 S and an extinction coefficient of 15.7 (E1%(1cm) 280 nm). Immunochemical methods were used to detect and quantitate PP3 in extracts of placentas and other human tissues. From one human term placenta an average of around 4 mg PP3 could be extracted; PP3 was also found to occur in extracts of adult human stomach. In concentrated extracts of other human tissues and in human body fluids this protein could not be detected, at least not in concentrations higher than 2 mg/dl.

Isolation and characterization of two membrane-associated placental tissue proteins.

Two membrane associated placental tissue proteins (PP4 and MP1) have been isolated and characterized. Both proteins are found in the soluble as well as solubilized protein fractions of the human placenta and thus appear to be at least partly associated with placental membranes. PP4 has a molecular weight of 35000 and apparently consists of a single peptide chain. It has an electrophoretic mobility in between the alpha 1- and alpha 2-globulins, an isoelectric point of 4.85 and a sedimentation coefficient of 3.3 S. The carbohydrate content of PP4 amounts to 2.4%. MP1 was isolated from placental protein fractions solubilized with Triton X-100. It has a molecular weight of around 18000 and appears to be composed of two identical subunits which are non-covalently linked. MP1 was found to have an electrophoretic mobility in between the alpha 2- and beta 1-globulins, an isoelectric point of 4.75 and a sedimentation coefficient of 6.65 S. MP1 is a glycoprotein which contains 9.6% carbohydrates. Immunochemical methods were used to detect and quantitate PP4 and MP1 in extracts of placentae and other human tissues. MP1 appears to be specific to the placenta, whereas PP4 was found to occur also in certain other human tissues. The diagnostic significance of detection and measurement of these proteins in tissues and body fluids is presently under investigation.

Isolation and characterization of four new placental tissue proteins (PP18, PP19, PP20, PP21).

Four new soluble placental tissue proteins (PP18, PP19, PP20, PP21) have been isolated to purity from saline extracts of human term placentas. Two of the new proteins appear to be partly associated with placental membranes; they also could be detected in placental protein fractions obtained by extracting the insoluble part of the placental tissue with solubilizing agents after the soluble material had been removed by washing with saline. The new placental proteins were characterized by their physical properties as well as by their carbohydrate and aminoacid compositions. Specific antisera to the new proteins were obtained by immunizing animals with the corresponding purified proteins. They were used to detect and quantitate the new proteins in extracts of placentas and other human tissues by immunochemical methods. From one human term placenta an average of 2 mg PP18, 90 mg PP19, 0.5 mg PP20, and around 7 mg PP21 could be extracted. None of these new proteins is specific to the placenta; they also were found to occur in extracts of certain other human tissues. The immunohistochemical localization of these proteins as well as measurement of their concentrations in body fluids by sensitive radioimmunoassays are presently under investigation.

Purification and characterization of two new soluble placental tissue proteins (PP13 and PP17).

Two new soluble placental tissue proteins (PP13 and PP17) have been isolated and characterized. PP13 has an electrophoretic mobility the same as that of albumin, an isoelectric point in the range 4.7-4.8 and a sedimentation coefficient of 3.1 S. Its molecular weight was found to be 30 000. PP13 appears to be composed of two identical subunits which are held together by disulfide bonds. PP17 has an electrophoretic mobility in between the beta 1- and alpha 2-globulins, an isoelectric point in the range 5.2-5.3 and a sedimentation coefficient of 2.7 S. Its molecular weight was determined to be 30 300 by ultracentrifugation and 38 000 by SDS-polyacrylamide gel electrophoresis. PP17 apparently consists of a single peptide chain. The amino acid and carbohydrate compositions of these proteins also have been determined. Immunochemical methods were used to detect and quantitate the new proteins in extracts of placental and other human tissues as well as in body fluids. From one human term placenta an average of 3.7 mg PP13 and 2.5 mg PP17 could be extracted. In concentrated extracts of other human tissues and in body fluids, these proteins could not be detected, at least not in concentrations higher than 1 mg/dl. The immunohistochemical localization of these proteins as well as measurement of their concentrations in body fluids by sensitive radioimmunoassays are presently under investigation.

[Isolation and characterization of the placental protein pp5 (author's transl)]. / Isolierung und Charakterisierung des Plazenta-Proteins PP5.

The isolation and characterization of placemental protein PP5 is described. The purification was achieved by use of immunoadsorbents. From the tissue of one human term placenta an average amount of 15 mg PP5 can be extracted. PP5 apparently is specific for the placenta; it could not be detected in extracts from other human tissues. In sera from pregnant women PP5 is not present or only in trace amounts (less than 0.1 mg%). In the ultracentrifuge PP5 was found to have a sedimentation coefficient of 2.8 S and a molecular weight of 36,600 daltons. Electrophoretically the protein migrates as a fast beta1-globulin. The isoelectric point was determined to be 4.6. PP5 is a glycoprotein and contains 19.8% carbohydrates (hexoses 10.0%, hexosamine 4.4%, fucose 0.4%, sialic acid 5.0%). The amino acid composition of the protein is reported, too. PP5 was found to inhibit the activity of trypsin and plasmin; the biological role of this protein therefore may be the inhibition of proteases.

[Isolation and characterization of a new tissue protein (pp7) from human placentae (author's transl)]. / Isolierung und Charakterisierung eines neuen Gewebsproteins (PP7) aus menschlichen Plazenten

The isolation and characterization of a new tissue protein (PP7) from human placentae is described. The placental protein PP7 is not specific for the placenta; it is found in relative high concentrations in other human tissues as well. Trace amounts of PP7 occur in erythrocytes, too. In sera this protein could not be detected with the gel diffusion test of Ouchterlony nor with the electroimmunoassay technique. PP7 has a molecular weight of around 40,000 daltons and is composed of two identical subunits which are non-covalently linked. In the ultracentrifuge PP7 sediments with 3.5 S. The isoelectric point was found to be 4.9. Electrophoretically the protein migrates in the range between alpha 2 and beta1-globulins. PP7 is a glycoprotein; its carbohydrate content amounts to 5.4% (hexoses 3.0%, Hexosamine 1.2%, Fucose 0.2%, sialic acid 1.0%). The amino acid composition of the protein is reported, too.

[Isolation and characterization of pregnancy associated alpha2 glycoprotein]. / Isolierung und Charakterisierung des schwangerschafts-assoziierten alpha2-Glykoproteins (alpha2-PAG)

A method using immunoadsorbents for the isolation of pregnancy-associated alpha2-glycoprotein (alpha2-PAG) from the extract of human placentae is described. The physical properties and the chemical composition of the purified protein are determined: alpha2PAG sediments with 11,5 S, has a molecular weight of 360 000 daltons and is composed of subunits having a molecular weight of 180000, which are held together by disulfide bonds. The isoelectric point was found to be pH 4,7 and the extinction coefficient (E1%1cm) was determined to be 9,7 at 277 nm. The carbohydrate content of the molecule amounts to 12,1% (hexose 6,0%, hexosamine 3,7%, fucose 0,06%, sialic acid 2,4%). An analysis of the amino acids is reported, too. The purified alpha2PAG was used to determine the absolute concentrations of this protein in a reference standard and in sera.