The genetic basis of pulmonary arterial hypertension

Pulmonary arterial hypertension (PAH) is a rare disease characterized by distinctive changes in pulmonary arterioles that lead to progressive elevation of pulmonary artery pressure, pulmonary vascular resistance, right ventricular failure, and a high mortality rate. The etiology of PAH is heterogeneous and incompletely understood. Based on clinical classification, WHO Group 1 PAH includes sporadic disease (idiopathic PAH), inherited PAH (heritable PAH), and association with certain medical conditions (associated PAH). Genes play an important role in idiopathic and heritable PAH. Mutations in bone morphogenetic protein receptor 2 (BMPR2), a member of the transforming growth factor β (TGFβ) superfamily of receptors, have been identified in 70 % of cases of familial PAH, as well as in 10–40 % of cases of idiopathic PAH. Mutations in ALK-1, ENG, SMAD4 and SMAD8, other TGFβ family members, are additional rare causes of PAH. CAV1 regulates SMAD2/3 phosphorylation, and mutations in CAV1 are a rare cause of PAH. KCNK3 is a member of the two-pore domain potassium channels expressed in pulmonary artery smooth muscle cells, and mutations in KCNK3 are a rare cause of both familial and IPAH. The genetics of PAH are complex due to incomplete penetrance and genetic heterogeneity. In addition to rare mutations as a monogenic cause of HPAH, common variants in cerebellin 2 (CBLN2) increase the risk of PAH by approximately twofold. PAH in children is much more heterogeneous than in adults and can be associated with several genetic syndromes, specifically syndromes with congenital heart disease, vascular disease, and hepatic disease. Clinical genetic testing is available for PAH and should be considered in families to allow for more definitive risk stratification and allow for reproductive planning.     

A European multicenter study of phenylalanine hydroxylase deficiency: classification of 105 mutations and a general system for genotype-based prediction of metabolic phenotype.

Phenylketonuria (PKU) and mild hyperphenylalaninemia (MHP) are allelic disorders caused by mutations in the gene encoding phenylalanine hydroxylase (PAH). Previous studies have suggested that the highly variable metabolic phenotypes of PAH deficiency correlate with PAH genotypes. We identified both causative mutations in 686 patients from seven European centers. On the basis of the phenotypic characteristics of 297 functionally hemizygous patients, 105 of the mutations were assigned to one of four arbitrary phenotype categories. We proposed and tested a simple model for correlation between genotype and phenotypic outcome. The observed phenotype matched the predicted phenotype in 79% of the cases, and in only 5 of 184 patients was the observed phenotype more than one category away from that expected. Among the seven contributing centers, the proportion of patients for whom the observed phenotype did not match the predicted phenotype was 4%-23% (P<.0001), suggesting that differences in methods used for mutation detection or phenotype classification may account for a considerable proportion of genotype-phenotype inconsistencies. Our data indicate that the PAH-mutation genotype is the main determinant of metabolic phenotype in most patients with PAH deficiency. In the present study, the classification of 105 PAH mutations may allow the prediction of the biochemical phenotype in >10,000 genotypes, which may be useful for the management of hyperphenylalaninemia in newborns.     

Scanning by DOVAM-S detects all unique sequence changes in blinded analyses: evidence that the scanning conditions are generic

The [detection of virtually all mutations]-SSCP (DOVAM-S) is a highly sensitive variant of single strand conformation polymorphism (SSCP). Mutations in the factor IX gene were used to find a set of five SSCP conditions that detects virtually all mutations. A blinded analysis of the factor IX gene in patients with hemophilia B detected 82 of 82 unique mutations. Since the method was developed and tested on the factor IX gene, it is possible that the conditions selected work more efficiently in the factor IX gene than in other genes. To test the general applicability of the conditions under which DOVAM-S detected all mutations in this gene, blinded analyses were performed in the human factor VIII and ataxia-telangiectasia (ATM) genes. Segments were amplified individually, combined into groups of 16 to 18 amplified segments and electrophoresed in five different nondenaturing conditions of varying matrices, buffers, temperatures and additives. Blinded analyses were performed in 92 samples from patients with hemophilia A (factor VIII gene) and 19 samples from A-T patients (ATM gene). Combined with an earlier blinded analysis in the factor IX gene, all of the 250 mutations and polymorphisms (180 of which are unique) were detected in both analyses. For two, three and four joint conditions, the average detection frequency ranged from 77%-97%, 91%-100% and 95%-100%, respectively. For each of the genes, one mutation may have been missed if only four conditions were used. With DOVAM-S, approximately 500 kb of autosomal sequence can be scanned in five gels with virtually 100% detection of mutations within the scanned region. The detection of 180 out of 180 unique sequence changes implies that DOVAM-S detects at least 96.5% (P = 0.03) of mutations. Blinded analyses that detect 400 unique sequence changes are required to determine that a scanning method detects at least 98.5% of mutations.     

Functional Changes in Pulmonary Arterial Endothelial Cells Associated with BMPR2 Mutations

Pulmonary arterial hypertension (PAH) is a devastating disease characterized by abnormal remodeling of small, peripheral pulmonary arteries. Germline mutations in the bone morphogenetic protein receptor type 2 (BMPR2) gene are a major risk factor for developing PAH. At present, the correlation between the BMPR2 mutation and the patient''s prognosis remains controversial despite several investigations. In this study, we explored the functional effects of four BMPR2 mutations to dissect the functional significance of the BMPR2 gene defect. Cellular immunofluorescence assay of four mutants (Tyr67Cys, Thr268fs, Ser863Asn, and Gln433X) revealed that the BMPR2 protein containing Thr268fs, Ser863Asn, or Gln433X exhibited abnormal subcellular localization. The BrdU incorporation and TUNEL assay suggested that any of the BMPR2 mutations Thr268fs, Ser863Asn, or Gln433X could improve endothelial cell apoptosis and decrease cell proliferation. All of the four mutants could inhibit nitric oxide (NO) synthesis in HLMVE cells, and ET-1 levels increased in the cells transfected with mutant Ser863Asn. Our results will improve the understanding of the genotype-phenotype correlations and mechanisms associated with BMPR2 mutations.     

苯丙氨酸羟化酶（PAH）基因外显子7及其两侧内含子的突变研究

为探讨中国苯丙酮尿症（PKU）人群中苯丙氨酸羟化酶（PAH）基因外显子7的突变特征,对147例PKU患儿的294个PAH基因外显子7以及两侧部分内含子序列,应用PCR－单链构象多态性（SSCP）分析及基因序列分析的方法进行了筛查和确定。共发现13种突变基因：G239D、R241C、R241fs、R243Q、G247S、G247V、R252Q、L255S、R261Q、M276K、E280G、P281L、Ivs7+2T>A,其中7 种突变基因在中国PKU人群首次发现：G239D 、R241fs 、G247S 、E280G、L255S、R261Q、P281L,前4种在国际上尚未见到报道,并已提交到国际PAH突变数据库（www.pahdb.mcgill.ca）。突变基因的总频率为30.61%（90 /294）。突变涉及了错义、缺失、移码和剪接位点4种突变类型。结果明确了PAH基因外显子7的突变种类和分布等特征,表明外显子7是中国人PAH基因突变的热点区域。 Abstract: To study mutation in exon 7 of the gene for the phenylalanine hydroxylase（PAH）, the mutations in exon 7 and flanking sequence of PAH gene were detected by means of SSCP analysis and DNA sequencing, in 147 unrelated Chinese children with phynelketonuria and their parents. Thirteen different mutations, including 11 missense, 1 deletion and 1 splice mutation, were revealed in 90/294 mutant alleles (30.61%). The prevalent mutations were R243Q (22.8%) and Ivs7nt2t->a (2.38%). Seven novel mutations were identified: G239D, R241fsdelG, G247S, E280G, L255S, R261Q, P281L. These new mutations have not been described in Chinese PKU population and the first 4 mutants have not been reported and thus been submitted to www.pahdb,mcgill.ca. The missense was the most common type. The deletion and frameshift mutations were detected for the first time in Chinese PKU population. This study showed the mutation characteristics and their distribution in exon 7 of PAH gene and proved that the exon 7 was the hot region of PAH gene mutation in Chinese PKU population .     

The inference of phased haplotypes for the immunoglobulin H chain V region gene loci by analysis of VDJ gene rearrangements

The existence of many highly similar genes in the lymphocyte receptor gene loci makes them difficult to investigate, and the determination of phased "haplotypes" has been particularly problematic. However, V(D)J gene rearrangements provide an opportunity to infer the association of Ig genes along the chromosomes. The chromosomal distribution of H chain genes in an Ig genotype can be inferred through analysis of VDJ rearrangements in individuals who are heterozygous at points within the IGH locus. We analyzed VDJ rearrangements from 44 individuals for whom sufficient unique rearrangements were available to allow comprehensive genotyping. Nine individuals were identified who were heterozygous at the IGHJ6 locus and for whom sufficient suitable VDJ rearrangements were available to allow comprehensive haplotyping. Each of the 18 resulting IGHV│IGHD│IGHJ haplotypes was unique. Apparent deletion polymorphisms were seen that involved as many as four contiguous, functional IGHV genes. Two deletion polymorphisms involving multiple contiguous IGHD genes were also inferred. Three previously unidentified gene duplications were detected, where two sequences recognized as allelic variants of a single gene were both inferred to be on a single chromosome. Phased genomic data brings clarity to the study of the contribution of each gene to the available repertoire of rearranged VDJ genes. Analysis of rearrangement frequencies suggests that particular genes may have substantially different yet predictable propensities for rearrangement within different haplotypes. Together with data highlighting the extent of haplotypic variation within the population, this suggests that there may be substantial variability in the available Ab repertoires of different individuals.     

Single-strand conformation polymorphism for detection of mutations and base substitutions in phenylketonuria.

In the past few years, more than 20 different mutations have been reported in hyperphenylalaninemias. In southwestern Europe and Mediterranean countries, however, the mutant genotypes reported account for only a fraction (27%) of all mutant alleles at the phenylalanine hydroxylase (PAH) locus, and most of the mutations causing the disease remain unknown. In order to develop a strategy for rapid detection of mutation-containing exons, we applied the single-strand conformation-polymorphism (SSCP) technique to exons 3, 5, 7, and 12 of the PAH gene. We observed five abnormal patterns of migration in mutant PAH genes, and we consistently found base substitutions in the corresponding exons, with no false-positive results. By this procedure, two novel putative mutations were detected in the seventh exon of the PAH gene, (A259V and Y277D) and we were able to demonstrate that the delta I94, R158Q, R408W, and E280K mutations were easily detectable by the SSCP technique. This procedure is therefore of particular interest for rapid detection of mutation-containing exons and for determination of further genotype-phenotype correlations in hyperphenylalaninemias.     

Genetic heterogeneity in five Italian regions: analysis of PAH mutations and minihaplotypes

Molecular analysis of 289 chromosomes has been performed in a cohort of phenylketonuria (PKU) patients whose ancestors lived in five Italian regions, Calabria, Campania, Piemonte, Puglia/Basilicata and Sicilia. Phenylalaninehydroxylase (PAH) gene mutations and minihaplotypes (combinations of PAH gene STR and VNTR systems) have been determined for 78.5 and 64%, respectively, of the chromosomes studied. 21 different minihaplotypes and 24 PKU mutations were found. Heterogeneity tests carried out for the frequencies of mutations and minihaplotypes show that the distribution of eight mutations and four minihaplotypes is statistically heterogeneous in the five Italian regions. Although the evolutionary rate of microsatellites or the age of these mutations is difficult to estimate with accuracy, our findings taken together show a genetic stratification of the Italian population. These results rule out allelic homogeneity of PKU at the molecular level between regions of Italy, yet minihaplotype data may be of practical use for a multistep approach to PAH gene genotyping.     

Spectrum of phenylketonuria mutations in Western Europe and North Africa,and their relation to polymorphic DNA haplotypes at the phenylalanine hydroxylase locus

Summary A total of 252 chromosomes from 126 patients with phenylalanine hydroxylase (PAH) deficiencies were analyzed for both mutant genotypes and restriction fragment length polymorphism (RFLP) haplotypes at the PAH locus. The mutant genes studied originated either from Western Europe (116 alleles) or from Mediterranean countries (136 alleles). Only 27% of all mutant alleles were found to carry identified mutations, particularly mutations at codon 252 (2.3%), 261 (7.5%), 280 (6.3%), 408 (3.5%) and at the splice donor site of intron 12 (6.3%). The mutant genotypes were associated with RFLP haplotypes 7, 1, 38, 2 and 3 at the PAH locus respectively. Except for the splice mutation of intron 12, these associations were preferential, but not exclusive, since the other four mutations were found on the background of at least two RFLP haplotypes. These results, together with the observation that 85% of PAH deficient patients are heterozygotes for their mutant genotypes, emphasize the great heterogeneity of PAH deficiencies in Mediterranean countries and hamper systematic DNA testing for carrier status in this population.     

Identification of Two Genes Required in Tomato for Full Cf-9-Dependent Resistance to Cladosporium fulvum

Mutagenesis was used to identify and characterize plant genes required for fungal disease resistance gene function in tomato. Seed of a stock homozygous for the Cf-9 gene for resistance to Cladosporium fulvum were treated with ethyl methanesulfonate, and 568 M2 families were screened for mutations to C. fulvum sensitivity. Eight mutants with reduced resistance were isolated. Four mutations, all of which mapped to the Cf-9 gene, lost both resistance and response to the race-specific AVR9 elicitor. The other four mutations partially lost resistance and response to the AVR9 elicitor. Cytological analysis revealed that a unique host cell staining pattern accompanied the reduced-resistance phenotype in three mutants. Two of the mutants with reduced resistance mapped to Cf-9, and two mapped to two distinct loci designated Rcr-1 and Rcr-2 (Required for Cladosporium resistance) that are unlinked to Cf-9.     

Analysis of Dominant Mutations Affecting Muscle Excitation in Caenorhabditis Elegans

We examined mutations that disrupt muscle activation in Caenorhabditis elegans. Fifteen of 17 of these genes were identified previously and we describe new mutations in three of them. We also describe mutations in two new genes, exp-3 and exp-4. We assessed the degree of defect in pharyngeal, body-wall, egg-laying, and enteric muscle activation in animals mutant for each gene. Mutations in all 17 genes are semidominant and, in cases that could be tested, appear to be gain-of-function. Based on their phenotypes, the genes fall into three broad categories: mutations in 11 genes cause defective muscle activation, mutations in four genes cause hyperactivated muscle, and mutations in two genes cause defective activation in some muscle types and hyperactivation in others. In all testable cases, the mutations blocked response to pharmacological activators of egg laying, but did not block muscle activation by irradiation with a laser microbeam. The data suggest that these mutations affect muscle excitation, but not the capacity of the muscle fibers to contract. For most of the genes, apparent loss-of-function mutants have a grossly wild-type phenotype. These observations suggest that there is a large group of genes that function in muscle excitation that can be identified primarily by dominant mutations.     

Structural and functional analysis of the goat epsilon-globin genes

Since none of the vertebrate beta-globin loci studied to date has more than two functional embryonic beta-like globin genes, it would be unique if all six goat embryonic beta-globin genes were required for its survival. In this study we have asked whether all six embryonic genes in the goat are functional. This question has been addressed by examining the transient expression of these genes in HeLa cells and correlating these results with the sequence information obtained to date. Our studies show that only epsilon I and epsilon II are functional while the remaining four epsilon-globin genes are nonfunctional, i.e., pseudogenes. Interestingly, the two active epsilon-globin genes are located at the 5' end of the locus. While this unusual inactivation pattern may be the result of chance, it could also have resulted because the two duplication events, of the ancestral gene set epsilon-epsilon-psi beta-beta, did not include distally located regulatory region(s) essential for epsilon-globin gene expression. Once separated from the 5'-regulatory sequences the remaining four embryonic genes (epsilon III, epsilon IV, epsilon V and epsilon VI) accumulated mutations and became pseudogenes.     

Phenylketonuria missense mutations in the Mediterranean.

Two missense mutations have been identified in the phenylalanine hydroxylase (PAH) genes of an Italian phenylketonuria (PKU) patient. Both mutations occurred in exon 7 of the PAH gene, resulting in the substitution of Trp for Arg at amino acid 252 (R252W) and of Leu for Pro (P281L) at amino acid 281 of the protein. Expression vectors containing either the normal human PAH cDNA or mutant cDNAs were constructed and transfected into cultured mammalian cells. Extracts from cells transfected with either mutant construct showed negligible enzyme activity and undetectable levels of immunoreactive PAH protein as compared to the normal construct. These results are compatible with the severe classical PKU phenotype observed in this patient. Population genetic studies in the Italian population revealed that both the R252W and the P281L mutations are in linkage disequilibrium with mutant restriction fragment length polymorphism (RFLP) haplotype 1, which is the most prevalent RFLP haplotype in this population. The R252W mutation is present in 10% and the P281L mutation is present in 20% of haplotype 1 mutant chromosomes. These mutations are both very rare among other European populations, suggesting a Mediterranean origin for these mutant chromosomes.     

Highlights of HRCT imaging in IPF

Background

Aim of this prospective study was to compare clinical and genetic findings in children with idiopathic or heritable pulmonary arterial hypertension (I/HPAH) with children affected with congenital heart defects associated PAH (CHD-APAH).

Methods

Prospectively included were 40 consecutive children with invasively diagnosed I/HPAH or CHD-APAH and 117 relatives. Assessment of family members, pedigree analysis and systematic screening for mutations in TGFß genes were performed.

Results

Five mutations in the bone morphogenetic protein type II receptor (BMPR2) gene, 2 Activin A receptor type II-like kinase-1 (ACVRL1) mutations and one Endoglin (ENG) mutation were found in the 29 I/HPAH children. Two mutations in BMPR2 and one mutation in ACVRL1 and ENG, respectively, are described for the first time. In the 11 children with CHD-APAH one BMPR2 gene mutation and one Endoglin gene mutation were found. Clinical assessment of relatives revealed familial aggregation of the disease in 6 children with PAH (HPAH) and one CHD-APAH patient. Patients with mutations had a significantly lower PVR.

Conclusion

Mutations in different TGFß genes occurred in 8/29 (27.6%) I/HPAH patients and in 2/11 (18.2%) CHD-APAH patients and may influence the clinical status of the disease. Therefore, genetic analysis in children with PAH, especially in those with I/HPAH, may be of clinical relevance and shows the complexity of the genetic background.     

The Sin3p PAH domains provide separate functions repressing meiotic gene transcription in Saccharomyces cerevisiae

Meiotic genes in budding yeast are repressed during vegetative growth but are transiently induced during specific stages of meiosis. Sin3p represses the early meiotic gene (EMG) by bridging the DNA binding protein Ume6p to the histone deacetylase Rpd3p. Sin3p contains four paired amphipathic helix (PAH) domains, one of which (PAH3) is required for repressing several genes expressed during mitotic cell division. This report examines the roles of the PAH domains in mediating EMG repression during mitotic cell division and following meiotic induction. PAH2 and PAH3 are required for mitotic EMG repression, while electrophoretic mobility shift assays indicate that only PAH2 is required for stable Ume6p-promoter interaction. Unlike mitotic repression, reestablishing EMG repression following transient meiotic induction requires PAH3 and PAH4. In addition, the role of Sin3p in reestablishing repression is expanded to include additional loci that it does not control during vegetative growth. These findings indicate that mitotic and postinduction EMG repressions are mediated by two separate systems that utilize different Sin3p domains.     

Molecular characterization of minor gene rearrangements in Finnish patients with heterozygous familial hypercholesterolemia: identification of two common missense mutations (Gly823-->Asp and Leu380-->His) and eight rare mutations of the LDL receptor gene.

Two deletions of the low-density lipoprotein (LDL) receptor gene were previously shown to account for about two thirds of all mutations causing familial hypercholesterolemia (FH) in Finland. We screened the DNA samples from a cohort representing the remaining 30% of Finnish heterozygous FH patients by amplifying all the 18 exons of the receptor gene by PCR and searching for DNA variations with the SSCP technique. Ten novel mutations were identified, comprising two nonsense and seven missense mutations as well as one frameshift mutation caused by a 13-bp deletion. A single nucleotide change, substituting adenine for guanidine at position 2533 and resulting in an amino acid change of glycine to aspartic acid at codon 823, was found in DNA samples from 14 unrelated FH probands. This mutation (FH-Turku) affects the sequence encoding the putative basolateral sorting signal of the LDL receptor protein; however, the exact functional consequences of this mutation are yet to be examined. The FH-Turku gene and another point mutation (Leu380-->His or FH-Pori) together account for approximately 8% of the FH-causing genes in Finland and are particularly common among FH patients from the southwestern part of the country (combined, 30%). Primer-introduced restriction analysis was applied for convenient assay of the FH-Turku and FH-Pori point mutations. In conclusion, this paper demonstrates the unique genetic background of FH in Finland and describes a commonly occurring FH gene with a missense mutation closest to the C terminus thus far reported.     

Identification of three novel missense PKU mutations among Chinese.

Three novel missense mutations have been identified in the phenylalanine hydroxylase (PAH) genes of Chinese individuals afflicted with various degrees of phenylketonuria (PKU). A T-to-C transition was observed in exon 5 of the gene, resulting in the substitution of Phe161 by Ser161. Two substitutions, G-to-T and T-to-G, were observed in exon 7, resulting in the substitution of Gly247 by Val247 and Leu255 by Val255, respectively. Expression analysis demonstrated that these mutant proteins produced between 0 and 15% of normal PAH enzyme activity. Population screening of a Chinese sample population indicates that these mutations are quite rare, together accounting for only about 4% of all PKU alleles among the Chinese. The P161S and G247V mutations were each present on a single PAH RFLP haplotype 4 chromosome in patients form Northern China, while the L255V mutation was present on chromosomes of both haplotypes 18 and 21 in patients from Southern China. These results suggest that the remaining 30% of uncharacterized PKU alleles in the Chinese population may bear a large number of relatively rare PAH mutations.     

PAH 399 GTA(Val)→GTT(Val), a new silent mutation found in the Chinese

Summary A silent mutation or sequence polymorphism, an A to T substitution at codon 399 in exon 11 of the phenylalanine hydroxylase (PAH) gene has been identified by DNA sequence analysis in the Chinese. The frequencies of this new mutation in normal and abnormal (phenylketonuria; PKU) genes are 0.005 and 0.09, respectively, based on the analyses of 100 apparently normal individuals and 39 PKU patients, as demonstrated by DNA amplification with polymerase chain reaction (PCR) and oligonucleotide hybridization methods. The results suggest that there is linkage disequilibrium between this polymorphism and PKU mutations in the PAH gene; approximately 10% of defect PAH alleles in the Chinese population may be identified with this sequence polymorphic marker.     

Results of and prospects for methods of determination of the frequency of mutant cells for glycophorin A and T-cell receptor loci to estimate long-term genotoxic effect of ionizing radiation

The usage of two methods for assessment of somatic gene mutations for the purpose of biological dosimetry and estimation of consequences of irradiation at long time after the exposure has been discussed. The determination of cells bearing mutated glycophorin A (GPA) locus is a reliable method for biodosimetry at both short and long time after the acute irradiation according to our results and the data of other authors. For prolonged exposure, the GPA-method is less informative than in cases of acute irradiation. The determination of mutant cell frequency at T-cell receptor (TCR) locus may be used only at short time after the irradiation. Meanwhile, the TCR-method is more sensitive and informative for biodosimetry of recent irradiation than the GPA test. The both methods may be used for individual assessment of long-term health consequences after the irradiation, because persons with elevated frequencies of mutant cells may represent a group at high risk in respect to oncological diseases.