X Chromosome Research Articles

Effect of long noncoding RNA on retinal development and ocular diseases

Long noncoding RNAs (lncRNAs) are non-protein coding transcript larger than 200 nucleotides, making up a large fraction of cellular transcripts in mammalian.Recent years have seen a surge of studies aimed at functionally characterizing the role of lncRNAs in development and disease.It has been proved that this kind of noncoding RNAs regulate gene expression at epigenetic, transcriptional, and post-transcriptional levels.Retinal development is dependent on an accurately functioning network of transcriptional and translational regulators.Among the diverse classes of molecules involved, lncRNAs play a significant role.LncRNAs are known or suspected to play a functional role in the specification and maturation of retinal cell subtypes and involved in several ocular related diseases.Though the molecular mechanism of most lncRNAs is still largely unclear, it is likely that these molecules represent a major component involved in retinal cell fate determination.In the process of retinal development, lncRNAs could induce cell differentiation, affect cell cycle and regulate X chromosome inactivation.lncRNAs also play an important role in ocular diseases, such as anophthalmia, diabetic retinopathy and spinocerebellar movement disorder type 7.This manuscript reviewed recent advances in the role of lncRNAs in retinal development and ocular diseases, and the existing problems, which is of great significance for basic and clinical research and the development of new therapeutic target. Key words: Long noncoding RNAs; Retina development; Ocular diseases

Association of AGTR1 and ACE2 gene polymorphisms with structural atrial fibrillation in a Chinese Han population.

The renin-angiotensin system (RAS) is thought to play an important role in atrial fibrillation (AF). The RAS contains the ACE/AngII/AGTR1 axis and the ACE2/Ang(1-7)/MAS axis, which restrict each other via mutual antagonism and regulate myocardial hypertrophy, fibrosis and remodelling. The aim of our study was to investigate the association between single nucleotide polymorphisms (SNPs) in angiotensin-II type-1 receptor (AGTR1) and angiotensin-converting enzyme 2 (ACE2) and structural AF in a Chinese Han population. The SNPs (rs1492100, rs1492099, rs1492097, rs3772616) in AGTR1 and the SNP rs6632677 in ACE2 were compared in 300 structural AF patients (67.61±12.56 years) and 300 controls (66.08±12.47 years). The genotype frequencies of SNP rs1492099 in AGTR1 in the structural AF cohort vs controls were as follows: GG, 72.7 vs 83.0%; AG 26.0 vs 16.3%; AA 1.3 vs 0.7% (P=0.009). The frequency of the minor allele of SNP rs1492099 in AGTR1 was 14.2% in the structural AF group compared with 8.8% in the controls (t=0.004; odds ratio [OR], 1.727; 95% confidence interval [CI]: 1.154-2.487). In addition, the genotype frequencies of SNP rs6632677 in ACE2 in the structural AF male patients vs male controls were as follows: GG, 70.5 vs 83.1%; CG 26.3 vs 15.6%; and CC 3.2 vs 1.3% (P=0.029). The frequency of the minor allele of SNP rs6632677 in ACE2 was 16.3% in structural AF male patients compared with 9.1% in male controls (P=0.008; OR, 1.954; 95%CI: 1.196-3.192). Furthermore, we found an interaction between the SNP rs6632677 in ACE2 and the SNPs (rs1492100/rs1492099/rs3772616) in AGTR1 in structural AF patients by the multifactor dimensionality reduction (MDR) method. The results indicate that polymorphism rs1492099 in the AGTR1 gene is associated with structural AF in a Chinese Han population. It was hypothesized that the ACE2 gene, which maps to the X chromosome, may be correlated with the risk of structural AF in a Chinese Han male population. Furthermore, we found an interaction between ACE2 and AGTR1 in structural AF patients in a Chinese Han population.

High enhancer of zeste homolog 2 correlates with poor prognosis of estrogen receptor-positive breast cancers

Objective To observe the biological behaviors of breast cancer expressing defferent levels of histone 3 lysine 27 triple—methylation (H3K27me3), enhancer of zeste homolog 2 (EZH2) and ubiquitously transcribed tetratricopeptide repeat on chromosome X (UTX). Methods The expression levels of H3K27me3, EZH2 and UTX in 146 cases of breast cancer were detected and their prognostic significance was explored. Results H3K27me3 levels were higher in human epidermal growth factor receptor-2 (Her-2)-negative samples. EZH2 expression was higher in breast cancers with lymph node metastasis, diameter >20 mm, and have higher tumor grade and stage. Using a Cox regression model, H3K27me3 and EZH2 levels were independent prognostic factors for overall survival for all the breast cancers [Hazard ratio (HR)=2.57, 95% confidence interval (CI): 1.63-4.04, P<0.01] even the estrogen receptor (ER)-positive subgroup (HR=2.78, 95%CI: 1.52-5.09, P<0.01) in our study. The combination of low H3K27me3 (HR=0.18, 95%CI: 0.07-0.49, P<0.01) and high EZH2 (HR=3.34, 95%CI: 1.20-9.27, P<0.05) was significantly associated with worse survival. UTX level was not significantly associated with prognosis and there were no correlations between H3K27me3 and EZH2/UTX expression. Conclusion Low H3K27me3 and high EZH2 can be used as a prognostic factor for shorter overall survival for breast cancer, including the ER+ breast cancer subtype. Key words: Breast cancer; Histone modification; Enhancer of zeste homolog 2; Prognosis

Sex-linked markers in the North American green frog (Rana clamitans) developed using DArTseq provide early insight into sex chromosome evolution.

BackgroundThe extent to which sex reversal is associated with transitions in sex determining systems (XX-XY, ZZ-ZW, etc.) or abnormal sexual differentiation is predominantly unexplored in amphibians. This is in large part because most amphibian taxa have homomorphic sex chromosomes, which has traditionally made it challenging to identify discordance between phenotypic and genetic sex in amphibians, despite all amphibians having a genetic component to sex determination. Recent advances in molecular techniques such as genome complexity reduction and high throughput sequencing present a valuable avenue for furthering our understanding of sex determination in amphibians and other taxa with homomorphic sex chromosomes like many fish and reptiles.ResultsWe use DArTseq as a novel approach to identify sex-linked markers in the North American green frog (Rana clamitans melanota) using lab-reared tadpoles as well as wild-caught adults from seven ponds either in undeveloped, forested habitats or suburban ponds known to be subject to contamination by anthropogenic chemicals. The DArTseq methodology identified 13 sex-linked SNP loci and eight presence-absence loci associated with males, indicating an XX-XY system. Both alleles from a single locus show partial high sequence homology to Dmrt1, a gene linked to sex determination and differentiation throughout Metazoa. Two other loci have sequence similarities to regions of the chimpanzee and human X-chromosome as well as the chicken Z-chromosome. Several loci also show geographic variation in sex-linkage, possibly indicating sex chromosome recombination. While all loci are statistically sex-linked, they show varying degrees of female heterozygosity and male homozygosity, providing further evidence that some markers are on regions of the sex chromosomes undergoing higher rates of recombination and therefore further apart from the putative sex determining locus.ConclusionThe ease of the DArTseq platform provides a useful avenue for future research on sex reversal and sex chromosome evolution in vertebrates, particularly for non-model species with homomorphic or cryptic or nascent sex chromosomes.Electronic supplementary materialThe online version of this article (doi:10.1186/s12864-016-3209-x) contains supplementary material, which is available to authorized users.

X-linked dominant protoporphyria: report of a pedigree and detection of ALAS2 gene mutations

Objective To report a pedigree with X-linked dominant protoporphyria (XLDPP), and to detect 5-aminolevulinic acid synthetase 2 (ALAS2) gene mutations in this pedigree. Methods A clinical investigation was performed in a pedigree with XLDPP, and relevant data were collected from family members. A next-generation sequencing method was applied to screen possible mutation sites, and Sanger sequencing was performed to determine pathogenic gene mutations. Dermoscopy was conducted to observe skin lesions in the patients with XLDPP, and the Fotofinder system and very high frequency (VHF) ultrasound system were utilized to assess the severity of photodamage. Liver and gallbladder ultrasonography as well as blood examination were performed for all the family members. Results A deletion mutation, c.1706-1709 ΔAGTG, was detected in the ALAS2 gene on the X chromosomes of all the patients in this family, which led to replacement or loss of 19-20 C-terminal residues through transcriptional frameshifting, and eventually caused an increase in ALAS2 activity. In the patients with XLDPP, skin photodamage was relatively severe; protoporphyrin-induced hepatobiliary damage was observed and aggravated with age; anemia and iron deficiency occurred sometimes. Conclusion The deletion mutation c.1706-1709 ΔAGTG of the ALAS2 gene may be the underlying cause of XLDPP in this pedigree. Key words: Porphyria, erythropoietic; Genetic diseases, X-linked; DNA mutational analysis; 5-Aminolevulinic acid synthetase 2 gene; Photoaging

Chromosome Painting in Triatomine Insects Reveals Shared Sequences Between X Chromosomes and Autosomes.

In order to provide a broad picture on the origin and evolution of holocentric X chromosomes in heteropteran species, we prepared a sex chromosome painting probe by microdissection of the X1 and X2 chromosomes from a kissing bug Mepraia spinolai (Hemiptera: Reduviidae: Triatominae). Fluorescence in situ hybridization on four species of the Triatomini having different amounts of autosomal heterochromatin and sex chromosome systems show that the Xs probe hybridizes on the euchromatin, located both on autosomes and X chromosomes. The heterochromatic Y chromosome and autosomal heterochromatic regions always appear free of hybridization signals. The hybridization results of the Xs probe on Rhodnius prolixus (Rhodniini) is completely different to that observed in Triatomini species. The hybridization signals are small and scattered on all euchromatin, without specific regions including the X chromosome. These results are in accordance with previous data obtained by genomic in situ hybridization and fluorescent banding, suggesting a clear differentiation in the repeat sequence composition of both sex chromosomes between Triatomini and Rhodniini tribes. These results also support that each sex chromosome in Triatomini has evolved independently from different autosomal pairs of a common ancestor, as described in other insect orders.

A new multiple sex chromosome system X1X1X2X2/X1Y1X2Y2 in Siluriformes: cytogenetic characterization of Bunocephalus coracoideus (Aspredinidae).

We analyzed one Bunocephalus coracoideus population from the Negro River basin using cytogenetic techniques. The results showed a diploid number of 42 chromosomes in both sexes, with the karyotypic formula4m+14sm+24a and fundamental number (FN)=60 for females and the formula5m+14sm+23a and FN=61 for males, constituting an X1X1X2X2/X1Y1X2Y2 multiple sex chromosome system. The constitutive heterochromatin is distributed in the pericentromeric regions of most of the chromosomes, except for the sex chromosomes, of which the X1, X2, and Y1 chromosomes were euchromatic and the Y2 chromosome was partially heterochromatic. 18S rDNA mapping confirmed the presence of nucleolar organizer regions on the short arms of the fifth chromosomal pair for both sexes. The 5S rDNA is present in the terminal regions of the short arms on the 2nd, 10th, and 12th pairs and on the X2 chromosome of both sexes; however, we observed variations in the presence of these ribosomal cistrons on the Y1 chromosome, on which the cistrons are pericentromeric, and on the Y2 chromosome, on which these cistrons are present in the terminal portions of the short and long arms. Telomeric sequences are located in the terminal regions of all of the chromosomes, particularly conspicuous blocks on the 10th and 12th pairs and internal telomeric sequences in the centromeric regions of the 1st, 6th, and 9th pairs for both sexes. This work describes an new sex chromosomes system for the Siluriformes and increases our genetic knowledge of the Aspredinidae family.

First Report of Sex Chromosomes in Achiridae (Teleostei: Pleuronectiformes) with Inferences About the Origin of the Multiple X1X1X2X2/X1X2Y System and Dispersal of Ribosomal Genes in Achirus achirus.

American soles (family Achiridae) have been characterized by remarkable chromosomal variation even though several species lack basic cytogenetic information. This trend indicates that chromosomal traits can be useful to taxonomy once the morphological identification of some taxa in this family (e.g., Achirus species) is controversial. In this work, we expand the cytogenetic data in Achiridae by providing the first karyotypic analysis of Achirus achirus. An unusual multiple sex chromosome system (X1X1X2X2/X1X2Y) was detected, once males presented 2n = 33 with three unpaired chromosomes (X1X2Y) while females presented 2n = 34 with two acrocentric pairs (X1X1 and X2X2) found in hemizygosis in males. The 18S rDNA clusters were observed interspersed with GC-rich sites in a single pair. However, the 5S rRNA genes were dispersed through the genome of A. achirus in a sex-specific manner (10 clusters in males and 12 in females), as a result of the presence of ribosomal cistrons in X1 and X2 chromosomes. This pattern allowed us to infer that Y chromosome has evolved by partial deletion followed by fusion of proto-X1 and proto-X2 homologous chromosomes. The high rate of genomic evolution in Achiridae could have favored their reproductive isolation and speciation even in sympatric conditions.

Human Xq28 inversion polymorphism: From sex linkage to Genomics--A genetic mother lode.

An inversion polymorphism of the filamin and emerin genes at the tip of the long arm of the human X-chromosome serves as the basis of an investigative laboratory in which students learn something new about their own genomes. Long, nearly identical inverted repeats flanking the filamin and emerin genes illustrate how repetitive elements can lead to alterations in genome structure (inversions) through nonallelic homologous recombination. The near identity of the inverted repeats is an example of concerted evolution through gene conversion. While the laboratory in its entirety is designed for college level genetics courses, portions of the laboratory are appropriate for courses at other levels. Because the polymorphism is on the X-chromosome, the laboratory can be used in introductory biology courses to enhance understanding of sex-linkage and to test for Hardy-Weinberg equilibrium in females. More advanced topics, such as chromosome interference, the molecular model for recombination, and inversion heterozygosity suppression of recombination can be explored in upper-level genetics and evolution courses. DNA isolation, restriction digests, ligation, long PCR, and iPCR provide experience with techniques in molecular biology. This investigative laboratory weaves together topics stretching from molecular genetics to cytogenetics and sex-linkage, population genetics and evolutionary genetics.

The cryptic Y-autosome translocation in the small Indian mongoose, Herpestes auropunctatus, revealed by molecular cytogenetic approaches.

In initial studies of the eutherian small Indian mongoose (Herpestes auropunctatus), the Y chromosome could not be identified in somatic cells. The male chromosome number is uniquely odd, 2n = 35, whereas that of females is 2n = 36. Previous reports indicated that this unique karyotype resulted from a translocation of the ancestral Y chromosome to an autosome. However, it has been difficult to identify the chromosomes that harbor the translocated Y chromosomal segment because it is an extremely small euchromatic region. Using a Southern blot analysis, we detected four conserved Y-linked genes, SRY, EIF2S3Y, KDM5D, and ZFY, in the male genome. We cloned homologues of these genes and determined their sequences, which showed high homology to genes in two carnivore species, cat and dog. To unambiguously identify the Y-bearing autosome, we performed immunostaining of pachytene spermatocytes using antibodies against SYCP3, γH2AX, and the centromere. We observed trivalent chromosomes, and the associations between the distal ends of the chromosomes were consistent with those of Y and X1 chromosomes. The centromere of the Y chromosome was located on the ancestral Y chromosomal segment. We mapped the complementary DNA (cDNA) clones of these genes to the male chromosomes using fluorescence in situ hybridization (FISH), and the linear localization of all genes was confirmed by two-colored FISH. These Y-linked genes were localized to the proximal region of the long arm of a single telomeric chromosome, and we successfully identified the chromosome harboring the ancestral Y chromosomal segment.

Distribution and Associations of GATA Repeats in Rice Genome

GATA repeats are associated with sex differentiation in man, buffalo, mouse and even in plants such as papaya. Human X-chromosome region Xp22 that escapes inactivation is ten-fold enriched in GATA repeats suggesting a role in preventing heterochromatinization. The close proximity of GATA repeats to matrix-associated regions (MARs) indicates a role in chromatin organization and function. Chromosome-wise distribution and density of GATA repeats, neighboring genes and Matrix associated regions were analyzed in rice. (GATA)3 and higher repeats were distributed non-randomly with the highest frequency on chromosome 11. About 60% of the repeats were found in intergenic regions flanked by regulatory genes involved in stress response or transposable elements. The GATA associated MAR sequences in rice had at least one or more of the consensus sequence to which GATA factors bind. The genomic milieu around GATA repeats suggests that their genomic contextmay determine their role in chromatin organization and gene regulation.

Correction: The Presence of the Y-Chromosome, Not the Absence of the Second X-Chromosome, Alters the mRNA Levels Stored in the Fully Grown XY Mouse Oocyte

[This corrects the article DOI: 10.1371/journal.pone.0040481.].

Genetic analysis and prenatal diagnosis of Xp deletion in a family with Duchenne/Becker muscular dystrophy

OBJECTIVE To delineate a deletional mutation of the Dystrophin gene on the short arm of chromosome X in a family affected with Duchenne/Becker muscular dystrophy. METHODS G-banded karyotyping, multiple ligation probe amplification (MLPA), array-based comparative genomic hybridization(array-CGH) and whole genome exon high-throughput sequencing were employed to delineate the mutation in the family. RESULTS GTG banding has demonstrated deletion of the terminal part of the short arm of chromosome X in the fetus. The same deletion was also found in its mother and maternal grandmother. MLPA analysis has revealed removal of exons 52 to 79 of the Dystrophin gene. A 30 Mb deletion in Xp22.33-p21.1 and a 10 Mb duplication in Xq27.2-q28 were identified by array-CGH and whole genome exon high-throughput sequencing. CONCLUSION The Xp deletion has led to deletion of exons 52 to 79 of the Dystrophin gene in the family. The female carriers also had certain features of Turner syndrome due to the same deletion.

AB048. X-chromosomal SNPs variation in populations of Russia

X-chromosome markers are informative tool for studying a genetic diversity in human populations and have become a useful in DNA identification when certain complex kinship cases need to be unravelled. In this work we present population genetic data on X-chromosome-wide SNPs in North Eurasian populations and report XSNP multiplex system for forensic genetics. A total of 2,867 X-chromosomal SNPs were genotyped in 12 populations using Illumina microarray platform. Twelve populations under study (Komi, Mordva, Russians, Kirghiz, Kazakh, Uzbek, Buryat, Yakut, Evenk, Tuva, Khanty, Ket) represent various language families and geographic regions of North Eurasia (Eastern Europe, Central Asia, Siberia and North Asia). North Eurasian populations are highly genetically differentiated with respect to XSNPs allele frequencies. Average level of genetic differentiation (Gst) for 12 populations is 6.03% and ranged from 1.05% to 30.05% per individual SNP. Principal component analysis of allele frequencies demonstrated geographic pattern of population clustering, as well as longitudinal gradient in genetic diversity. The 66 XSNPs characterized by high expected heterozygosity and linkage equilibrium in populations under study were selected for constructing a panel for forensic genetic applications. Average heterozygosity of selected SNPs varied from 0.4925 to 0.4958. Overall values of power of discrimination for males and females (PDm and PDf) obtained with these XSNPs set are several magnitude higher than those for standard forensic STR panels. Protocol for multiplex amplification of 66 XSNPs in two separate multiplex PCR reactions and MALDI-TOF mass spectrometry genotyping was developed. North Eurasian populations demonstrate high level of genetic diversity and differentiation for X-chromosome-wide SNPs. Based on obtained population genetic data, highly informative multiplex XSNPs panel for forensic genetics was developed.

Involvement of lncRNA dysregulation in gastric cancer.

Benefiting from the fast development of sequencing technique and bioinformatics methods, more and more new long non-coding RNAs (lncRNAs) are discovered and identified. lncRNAs were firstly thought to be transcription noise that from genome desert without biological function; however, as the discovery of lncRNA XIST and HOTAIR uncovers the emerging roles of lncRNAs in development and tumorigenesis. In the past decades, accumulating evidence have indicated that lncRNAs involve in a wide range of biological functions, such as X-chromosome inactivation, reprogramming stem cell pluripotency, regulation of the immune response and carcinogenesis. Although lots of studies have demonstrated that dysregulation of lncRNAs involve in diverse diseases including cancers, the underlying molecular mechanisms of lncRNAs are not well documented. Interestingly, our previous studies and others' have shown that numerous of lncRNAs expression was misregulated in gastric cancer. In this review, we will focus on the dysregulated lncRNAs and their biological function and underlying pathways or mechanisms in GC. Finally, we will discuss the potential roles of lncRNAs acting as biomarkers or therapeutic targets in GC patients.

CDKL5 in different atypical Rett syndrome variants: Description of the first eight patients from Spain

Mutations in cyclin-dependent kinase-like 5 (CDKL5) have been observed in patients with epileptic encephalopathies and atypical variants of Rett syndrome (RTT) associated with early epilepsy. Determination of the type and location ofCDKL5 mutations may provide molecular diagnosis and prognostic information and aid in genetic counseling for the family. Molecular analysis of CDKL5 and X-chromosome inactivation pattern in 53 Spanish RTT girls (without identifiable methyl-CpG-binding-protein 2) and nine boys with epileptic encephalopathy was performed. De novo CDKL5 mutations were identified in eight atypical RTT patients: one late regression; one preserved speech, one congenital variant with epilepsy onset at 3 years of age, and five patients. An addi- tional five patients with early-onset epilepsy. Seizure types and CDKL5 mutations were identified. Seizures types included infantile spasms or tonic seizures, and developing polymorphic seizures that were resistant to antiepileptic drugs. Electroencephalography records were abnormal, without characteristic pathologic pattern. Epilepsy control (total or partial) was achieved with valproate (four patients) or carbamazepine (three patients). Long-term outcome was variable, depending on type of mutation and epilepsy control. These arethe firsteight girlsfromSpainwithCDKL5mutations.Mutationswere identifiedinearlyepilepsy variantswithearlyhypo- tonia, but also in other atypical variants without a molecular diagnosis. This study highlights the importance of CDKL5 analysis in all atypicalRTT patientswithoutan identifiablemethyl-CpG-binding-protein2 mutation.Typeandlocation ofCDKL5mutationandthe resultant effect on protein appear to determine the severity of epilepsy.

Characterization of CpG sites that escape methylation on the inactive human X-chromosome

In many whole genome studies of gene expression or modified cytosines, data from probes localized to the X-chromosome are removed from analyses due to gender bias. Previously, we observed population differences in cytosine modifications between Caucasian and African lymphoblastoid cell lines (LCLs) on the autosomes using whole genome arrays to measure modified cytosines. DNA methylation plays a critical role in establishment and maintenance of X-chromosome inactivation in females. Therefore, we reasoned that by investigating cytosine modification patterns specifically on the X-chromosome, we could obtain valuable information about a chromosome that is often disregarded in genome-wide analyses. We investigated population differences in cytosine modification patterns along the X-chromosome between Caucasian and African LCLs and identified novel sites that escape methylation on the inactive X-chromosome (Xi) in females. We characterized the chromatin state of these loci by incorporating the extensive histone modification ChIP-seq data generated by ENCODE. To explore the relationship between DNA and histone modifications further, we hypothesized that BRD4, a protein that binds acetylated histones, could be preventing some sites from becoming de novo methylated. To test this, we treated 4 female LCLs with JQ1, a small molecule inhibitor of BRD4, but found that JQ1 treatment induced minor changes in cytosine modification levels, and the majority of sites escaping methylation on the Xi remained unmethylated. This suggests that other epigenetic mechanisms or transcription factors are likely playing a larger role in protecting these sites from de novo methylation on the Xi.

Genetics: Mutations underlying azoospermia identified on the X chromosome.

Genetics: Mutations underlying azoospermia identified on the X chromosome.

Of mice and cats (both calico): Mary F Lyon, FRS (1925-2014).

Of mice and cats (both calico): Mary F Lyon, FRS (1925-2014).

Complement-mediated haemolysis and the role of blood transfusion in paroxysmal nocturnal haemoglobinuria.

Paroxysmal nocturnal haemoglobinuria (PNH) is a rare form of acquired chronic haemolytic anaemia with unique characteristics. From a clinical point of view the classical triad is that of intravascular haemolysis, thrombosis and cytopenias1,2. From the point of view of pathogenesis, PNH can be regarded as a non-malignant clonal disorder, in which a mutation of the X-linked PIG-A gene in a haematopoietic stem cell affects the its mature progeny in the peripheral blood, including erythrocytes, leucocytes and platelets3: this progeny is, therefore, referred to as a PNH clone4. In PNH patients the PNH clone co-exists with qualitatively normal blood cells, although in many cases these are a minority5. PNH has no gender predilection and an estimated population frequency of between 1 and 10 per million. Molecular basis of paroxysmal nocturnal haemoglobinuria The first biochemical abnormality to be demonstrated in red cells from patients with PNH was a deficiency of the membrane protein acetylcholinesterase6. Subsequent studies showed that several more membrane proteins were missing or markedly decreased in the blood cells belonging to the PNH clone. From the serological point of view it is interesting that one of these (decay accelerating factor, see below) bears the Cromer blood groups7. In the 1980s it emerged that these deficient membrane proteins have one important feature in common: they are anchored to the cell surface through a glycosyl phosphatidyl inositol (GPI) molecule8,9. This suggested that the underlying biochemical abnormality would be in the GPI biosynthetic pathway, and it was indeed pinpointed at the step at which N-acetylglucosamine (NAcGlcN) is transferred onto phoshatidyl inositol10. Through expression cloning in a GPI-deficient cell line Taroh Kinoshita’s group identified the PIG-A (phosphatidyl inositol glycan complementation group A) gene as being defective in PNH cells11, and PIG-A somatic mutations were indeed identified in PNH patients12. PIG-A maps to the short arm of the X chromosome (band p22.1)13. The PIG-A product is a 484 amino acid protein which, with three other proteins, forms NAcGlcN transferase, the enzyme that catalyses the first step of the biosynthesis of the GPI anchor. Almost all the somatic mutations of the PIG-A gene identified so far in PNH patients are either frame-shift or nonsense mutations, only two are large deletions14,15. It should be noted that one of these inactivating mutations is sufficient to give rise on its own to the PNH phenotype because PIG-A is X-linked: as a result, only one allele is present in men, and only one allele is active in somatic cells in women. Very recently a PNH patient has been reported who had no PIG-A mutation: instead, she had a germ-line mutation of another gene (PIG-T) encoding a GPI biosynthetic enzyme, and a somatic mutation of the other PIG-T allele16. This patient provides a powerful confirmation of the notion that the critical biochemical failure in the PNH clone is in biosynthesis of the GPI anchor; in terms of frequency, this is likely to remain an exceptional situation17.