Background: The prevalence of Fabry disease (FD) in Chinese patients with hypertrophic cardiomyopathy (HCM) is unclear. We aimed to evaluate the prevalence, clinical characteristics, and outcomes of FD in Chinese patients with HCM. Methods: Of 217 patients with HCM, FD probands were screened by next-generation sequencing at Fuwai Hospital. Medi-cal data from a-galactosidase A activity, electrocardiography, echocardiography, coronary angiography, cardiac magnetic resonance, pathological examination, and follow up was analyzed. Results: Two FD probands were observed (0.93% of patients with HCM), both of which were diagnosed with symp-tomatic obstructive HCM at 49 years of age. One proband had a GLA mutation (c.887T>C [p.M296T]) with a late-onset cardiac variant, which was characterized by dual ventricular hypertrophy and conduction disease with a perma-nent pacemaker. The other patient had a GLA mutation (c.758T>C [p.I253T]) with a classic phenotype and dual ven-tricular hypertrophy, atrioventricular block, renal failure, and recurrent cerebral infarction. Both probands had late gadolinium enhancement mainly in the basal segment of the inferolateral wall. Follow up revealed no exertional symp-toms or outflow obstruction after surgical septal myectomy in the two probands, and stable renal function was observed after 6 months of migalastat therapy in the later one. A family study revealed six female carriers and three sudden cardiac deaths. Conclusions: FD is not uncommon in Chinese patients with HCM. Multiple organic involvement, dual ventricular hypertro-phy, and conduction disease provide clinical clues for suspected FD, and early genetic screening is necessary. Surgical septal myectomy and migalastat improve the long-term prognosis of patients with FD.

目的 探讨基因检测在汉族肥厚型心肌病(hypertrophic cardiomyopathy,HCM)患者中Fabry病的基因突变情况及家系筛查中的应用,并分析基因型与表型的关系.方法 应用半导体靶向二代测序平台筛查在阜外医院诊断为HCM的217例患者,应用Sanger测序验证先证者和家系内成员的GLA基因突变位点,收集GLA突变携带者的临床资料并进行基因型与表型关联分析.结果 发现2例男性Fabry病先证者(在HCM中占比0.93％).1例携带GLA基因错义突变c.887T＞C(p.M296T),表现为迟发心脏型Fabry病;对其一家四代中的25个家庭成员进行家系突变筛查,结果发现有4个女性杂合突变携带者,其中1个确诊为HCM.另l例携带GLA基因错义突变c.758T＞C(p.I253T),表现为经典型Fabry病,累及肾和神经系统.对其一家四代中的32个家系成员进行家系调查,发现2个女性杂合突变携带者和2个男性早发心脏性猝死.两例先证者经室间隔心肌切除术后梗阻解除,后者应用分子伴侣药物Migalastat治疗后肾功能稳定于31 ml/min.结论 首次发现Fabry病在汉族HCM中并不少见,基因检测有助于早期鉴别诊断及筛查家系内突变携带者.GLA c.758T＞C为恶性基因型,男性突变携带者猝死风险高危,室间隔心肌切除术和Migalastat有助于改善预后.

Familial acne inversa (AI) is an autoinflammatory disorder that affects hair follicles and is caused by loss-of-function mutations in γ-secretase component genes. We and other researchers showed that nicastrin (NCSTN) is the most frequently mutated gene in familial AI. In this study, we generated a keratin 5-Cre-driven epidermis-specific Ncstn conditional knockout mutant in mice. We determined that this mutant recapitulated the major phenotypes of AI, including hyperkeratosis of hair follicles and inflammation. In Ncstnflox/flox;K5-Cre mice, the IL-36a expression level markedly increased starting from postnatal day 0 (P0), and this increase occurred much earlier than those of TNF-α, IL-23A, IL-1β, and TLR4. RNA-Seq analysis indicated that Sprr2d, a member of the small proline-rich protein 2 family, in the skin tissues of the Ncstnflox/flox;K5-Cre mice was also upregulated on P0. Quantitative reverse-transcription polymerase chain reaction showed that other Sprr2 genes had a similar expression pattern. Our findings suggested that IL-36a might be a key inflammatory cytokine in the pathophysiology of AI and involved in the malfunction of the skin barrier in the pathogenesis of AI.

Marfan syndrome (MFS) is a systemic connective tissue disease principally affecting the ocular, skeletal and cardiovascular systems. This autosomal dominant disorder carries a prevalence of 1:3,000 to 1:5,000. This study aims to define the mutational spectrum of MFS related genes in Chinese patients and to establish genotype-phenotype correlations in MFS. Panel-based targeted next-generation sequencing was used to analyze the FBN1, TGFBR1 and TGFBR2 genes in 123 unrelated Chinese individuals with MFS or a related disease. Genotype-phenotype correlation analyses were performed in mutation-positive patients. The results showed that 97 cases/families (78.9%; 97/123) harbor at least one (likely) pathogenic mutation, most of which were in FBN1; four patients had TGFBR1/2 mutations; and one patient harbored a SMAD3 mutation. Three patients had two FBN1 mutations, and all patients showed classical MFS phenotypes. Patients with a dominant negative-FBN1 mutation had a higher prevalence of ectopia lentis (EL). Patients carrying a haploinsufficiency-FBN1 mutation tended to have aortic dissection without EL. This study extends the spectrum of genetic backgrounds of MFS and enriches our knowledge of genotype-phenotype correlations.

Background/Aims: Liddle syndrome (LS) is a rare autosomal dominant disease caused by mutations in genes coding for epithelial sodium channel (ENaC) subunits. The aim of this study was to identify the mutation responsible for the LS in an extended Chinese family. Methods: DNA samples from the proband with early-onset, treatment-resistant hypertension, and hypokalemia and 19 additional relatives were all sequenced for mutations in exon 13 of the β-ENaC and γ-ENaC genes, using amplification by polymerase chain reaction and direct DNA sequencing. Results: Genetic testing of exon 13 of SCNN1B revealed duplication of guanine into a string of 3 guanines located at codon 602. This frameshift mutation is predicted to generate a premature stop codon at position 607, resulting in truncated β-ENaC lacking the remaining 34 amino acids, including the crucial PY motif. Among a total of 9 participants with the identical mutation, different phenotypes were identified. Tailored treatment with amiloride was safe and effective in alleviating disease symptoms in LS. No mutation of SCNN1G was identified in any of the examined participants. Conclusions: We report here a family affected by LS harboring a frameshift mutation (c.1806dupG) with a premature stop codon deleting the PY motif of β-ENaC. Our study demonstrates that the earlier LS patients are diagnosed by genetic testing and treated with tailored medication, the greater the likelihood of preventing or minimizing complications in the vasculature and target organs.

Background: Desminopathy, a hereditary myofibrillar myopathy, mainly results from the desmin gene (DES) mutations. Desminopathy involves various phenotypes, mainly including different cardiomyopathies, skeletal myopathy, and arrhythmia. Combined with genotype, it helps us precisely diagnose and treat for desminopathy. Methods: Sanger sequencing was used to characterize DES variation, and then a minigene assay was used to verify the effect of splice-site mutation on pre-mRNA splicing. Phenotypes were analyzed based on clinical characteristics associated with desminopathy. Results: A splicing mutation (c.735+1G>T) in DES was detected in the proband. A minigene assay revealed skipping of the whole exon 3 and transcription of abnormal pre-mRNA lacking 32 codons. Another affected family member who carried the identical mutation, was identified with a novel phenotype of desminopathy, non-compaction of ventricular myocardium. There were 2 different phenotypes varied in cardiomyopathy and skeletal myopathy among the 2 patients, but no significant correlation between genotype and phenotype was identified. Conclusions: We reported a novel phenotype with a splicing mutation in DES, enlarging the spectrum of phenotype in desminopathy. Molecular studies of desminopathy should promote our understanding of its pathogenesis and provide a precise molecular diagnosis of this disorder, facilitating clinical prevention and treatment at an early stage.

Osteogenesis imperfecta (OI) is a rare hereditary skeletal dysplasia, characterized by recurrent fractures and bone deformity. This study presents a clinical characterization and mutation analysis of 668 patients, aiming to establish the mutation spectrum and to elucidate genotype-phenotype correlations in Chinese OI patients. We identified 274 sequence variants (230 in type I collagen encoding genes and 44 in noncollagen genes), including 102 novel variants, in 340 probands with a detection rate of 90%. Compared with 47 loss-of-function variants detected in COL1A1, neither nonsense nor frameshift variants were found in COL1A2 (p < 0.0001). The major cause of autosomal recessive OI was biallelic variants in WNT1 (56%, 20/36). It is noteworthy that three genomic rearrangements, including one gross deletion and one gross duplication in COL1A1 as well as one gross deletion in FKBP10, were detected in this study. Of ten individuals with glycine substitutions that lie towards the N-terminal end of the triple-helical region of the α1(I) chain, none exhibited hearing loss, suggesting a potential genotype-phenotype correlation. The findings in this study expanded the mutation spectrum and identified novel correlations between genotype and phenotype in Chinese OI patients.

BACKGROUND:Liddle syndrome is an autosomal dominant form of monogenic hypertension. Phenotypic variability makes it difficult to identify patients with Liddle syndrome, resulting in misdiagnosis and severe complications at early age.OBJECTIVES:To identify mutation in SCNN1B and SCNN1G genes in an adolescent with suspicious Liddle syndrome and his family members and to explore the screening target subjects of Liddle syndrome.METHODS:Genetic analysis of the C-terminus of SCNN1B and SCNN1G genes was conducted in an adolescent, with treatment-resistant hypertension and hypokalemia, who was suspected of having Liddle syndrome, and his family members. A Medline research of the reported cases with Liddle syndrome was also performed.RESULTS:A recurrent SCNN1B mutation, c.1853C>A (p.P618H), was detected in the 19-year-old male patient, and family screening identified five additional members who were heterozygous for the mutation. The diagnosis of Liddle syndrome was made in all affected individuals. Despite the phenotypic variability, a systematic review of 54 reported index cases revealed the early-onset hypertension, aged no more than 30 years, as a common feature.CONCLUSIONS:Genetic screening for Liddle syndrome should be considered in hypertensive subjects with early penetrance, maybe no more than 30 years, after exclusion of common secondary causes of hypertension.

Objective To elucidate the clinical characteristics and genotype-phenotype relationships of Chinese patients with neonatal diabetes mellitus (NDM) caused by forkhead box P3 (FOXP3) gene mutations (FOXP3-NDM).Methods A total of 50 suspected patients with NDM were recruited from Peking Union Medical College Hospital.After the detailed collection of clinical information,target sequencing of 21 known causative genes of NDM were performed for molecular genetic diagnosis.Results Three patients with FOXP3-NDM were identified,separately carrying FOXP3 p.R312H,p.V408M,and p.P133L hemizygous mutations.All these 3 patients showed simple permanent NDM,without other common manifestations of IPEX syndrome until 9-12 years old.And they only need insulin therapy.However,all the foreign reported cases presented multiple autoimmune diseases besides NDM,and needed immunosuppressive therapy,even with the same mutation.Conclusion This is the first report of Chinese FOXP3-NDM patients,which showed totally different clinical phenotypes from foreign reported cases.The underlying molecular mechanism remains to be further studied.

BACKGROUND:Cardiomyopathies are the most common clinical and genetic heterogeneity cardiac diseases, and genetic contribution in particular plays a major role in patients with primary cardiomyopathies. The aim of this study is to investigate cases of inherited cardiomyopathy (IC) for potential disease-causing mutations in 64 genes reported to be associated with IC.METHODS:A total of 110 independent cases or families diagnosed with various primary cardiomyopathies, including hypertrophic cardiomyopathy, dilated cardiomyopathy, restrictive cardiomyopathy, arrhythmogenic right ventricular cardiomyopathy, left ventricular non-compaction, and undefined cardiomyopathy, were collected after informed consent. A custom designed panel, including 64 genes, was screened using next generation sequencing on the Ion Torrent PGM platform. The best candidate disease-causing variants were verified by Sanger sequencing.RESULTS:A total of 78 variants in 73 patients were identified. After excluding the variants predicted to be benign and VUS, 26 pathogenic or likely pathogenic variants were verified in 26 probands (23.6%), including a homozygous variant in the SLC25A4 gene. Of these variants, 15 have been reported in the Human Gene Mutation Database or ClinVar database, while 11 are novel. The majority of variants were observed in the MYH7 (8/26) and MYBPC3 (6/26) gene. Titin (TTN) truncating mutations account for 13% in our dilated cardiomyopathy cases (3/23).CONCLUSIONS:This study provides an overview of the genetic aberrations in this cohort of Chinese IC patients and demonstrates the power of next generation sequencing in IC. Genetic results can provide precise clinical diagnosis and guidance regarding medical care for some individuals.

Liddle syndrome (LS), a monogenetic autosomal dominant disorder, is mainly characterized by early-onset hypertension and hypokalemia. Clinically, misdiagnosis or missing diagnosis is common, since clinical phenotypes of LS are variable and nonspecific. We report a family with misdiagnosis of primary aldosteronism (PA), but identify as LS with a pathogenic frameshift mutation of the epithelial sodium channel (ENaC) β subunit. DNA samples were collected from a 32-year-old proband and 31 other relatives in the same family. A designed panel including 41 genes associated with monogenic hypertension was screened using next-generation sequencing. The best candidate disease-causing variants were verified by Sanger sequencing. Genetic analysis of the proband revealed a novel frameshift mutation c.1838delC (p.Pro613Glnfs*675) in exon 13 of SCNN1B. This heterozygous mutation involved the deletion of a cytosine from a string of three consecutive cytosines located at codons 612 to 613 and resulted in deletion of the crucial PY motif and elongation of the β-ENaC protein. The identical mutation was also found in 12 affected family members. Amiloride was effective in alleviating LS for patients. There were no SCNN1A or SCNN1G mutations in this family. Our study emphasizes the importance of considering LS in the differential diagnosis of early-onset hypertension. The identification of a novel frameshift mutation of SCNN1B enriches the genetic spectrum of LS and has allowed treatment of this affected family to prevent severe complications.

Single nucleotide polymorphisms (SNPs) are closely related to genetic diseases, but current SNP detection methods, such as DNA microarrays that include tedious procedures and expensive, sophisticated instruments, are unable to perform rapid SNPs detection in clinical practice, especially for those multiple SNPs related to genetic diseases. In this study, we report a sensitive, low cost, and easy-to-use point-of-care testing (POCT) system formed by combining amplification refractory mutation system (ARMS) polymerase chain reaction with gold magnetic nanoparticles (GMNPs) and lateral flow assay (LFA) noted as the ARMS-LFA system, which allow us to use a uniform condition for multiple SNPs detection simultaneously. The genotyping results can be explained by a magnetic reader automatically or through visual interpretation according to the captured GMNPs probes on the test and control lines of the LFA device. The high sensitivity (the detection limit of 0.04 pg/μL with plasmid) and specificity of this testing system were found through genotyping seven pathogenic SNPs in phenylalanine hydroxylase gene ( PAH, the etiological factor of phenylketonuria). This system can also be applied in DNA quantification with a linear range from 0.02 to 2 pg/μL of plasmid. Furthermore, this ARMS-LFA system was applied to clinical trials for screening the seven pathogenic SNPs in PAH of 23 families including 69 individuals. The concordance rate of the genotyping results detected by the ARMS-LFA system was up to 97.8% compared with the DNA sequencing results. This method is a very promising POCT in the detection of multiple SNPs caused by genetic diseases.

A 59-year-old Chinese woman presenting with shortness of breath after exertion, pitting oedema of the lower extremities and Raynaud's phenomenon was evaluated. Strongly positive ANA and anti-Scl-70 antibodies were found in serologic tests. Echocardiogram showed enlarged right ventricle and atrium as well as severe systolic dysfunction of both ventricles but without pulmonary arterial hypertension. Cardiac magnetic resonance showed diffused late gadolinium enhancement in both ventricular walls. Electrocardiograph showed Epsilon waves in the right precordial leads V1 and V2, and >500 ventricular extrasystoles per 24 hours (Holter). The initial diagnosis was Systemic Sclerosis (SSc) with cardiac involvement, but considering the family history of heart failure, the gene scanning was performed which revealed a novel DSG2 missense mutation in this patient and her siblings. This patient was ultimately diagnosed as SSc complicated by Arrhythmogenic right ventricular cardiomyopathy (ARVC). This is the first case of Chinese patient with this overlap syndrome.

Objective We performed mutation screening in patients with epilepsy by using two benchtop next-generation sequencing platforms, Illumina MiSeq and Ion Torrent PGM.The same target capture system was used, together with standard reagents, software and hardware for each of the plat-forms.Here we compared their performance and discussed the advantages and disadvantages of each se -quencer, optimized the process of mutation screening and provided suggestions for sequencing platform se -lection.Methods The peripheral blood of 30 patients with epilepsy was collected after obtaining informed consent, and genomic DNA was extracted.Ion XpressTMPlus gDNA Fragment Library Preparation kit and Illumina TruSeq DNA LT sample prep kit were used for library preparation in 8 and 22 samples, respec-tively.Coding region sequences of 428 target genes were enriched by NimbleGen target capture system and sequenced either on Illumina MiSeq or Ion Torrent PGM.Output data from PGM were analyzed by Ion Torrent Suite 3.2 for alignment and variants extraction, while data from Miseq were analyzed by BWA-MEM and Samtools.Results Up to 12 samples could be sequenced on Illumina Miseq per run, with to-tal throughput more than 5 Gb, 137 x mean coverage sequencing depth, 96.4% capture efficiency rate on target regions and 2056 ±9.042 variants detected per sample, and the whole test period lasted for 6 days.Comparatively, up to 8 samples per 318 v2 chip could be sequenced on PGM, with total through-put about 1.4 Gb, 70 x mean coverage sequencing depth, 96.1% capture efficiency rate on target re-gions and 3028 ±678.9 variants detected per sample,and the test period was 6 days.Conclusion Targeted sequence capture combined with next generation sequencing could be an efficient tool for rapid mutation detection in epilepsy.All two benchtop next-generation sequencers evaluated here could generate usable sequences; however there are some differences between the qualities of their data.Ion Torrent PGM is more flexible and cost effective, while Illumina MiSeq is suitable for large sample size detection and with higher throughput and more accuracy.

BACKGROUND:Pediatric cataract is a clinically and genetically heterogeneous disease which is a significant cause of lifelong visual impairment and treatable blindness. Our study aims to investigate the genotype spectrum in a group of Chinese patients with pediatric cataract.METHODS:We enrolled 39 families with pediatric cataract from October 2015 to April 2016. DNA samples of the probands were analyzed by target next-generation sequencing. Variants were validated using Sanger sequencing in the probands and available family members.RESULTS:In our cohort of 39 cases with different types of pediatric cataract, 23 cases were found to harbor putative pathogenic variants in 15 genes: CRYAA, CRYBA1, CRYBA4, CRYBB1, CRYGC, CRYGD, MIP, GCNT2, IARS2, NHS, BCOR, BFSP2, FYCO1, MAF, and PAX6. The mutation detection rates in the familial and sporadic cases were 75 and 47.8%, respectively. Of the 23 causative variants, over half were novel.CONCLUSIONS:This is a rare report of systematic mutation screening analysis of pediatric cataract in a comparably large cohort of Chinese patients. Our observations enrich the mutation spectrum of pediatric cataract. Next-generation sequencing provides significant diagnostic information for pediatric cataract cases, especially when considering sporadic and subtle syndromal cases.

Objective To identify the mutations of pathogenic gene of familial hypertrophic cardiomyopathy (HCM) in China,and analyze the genotype-phenotype correlation. Methods A Chinese family with HCM was investigated. Targeted resequencing was performed in the proband based on a custom AmpliSeq panel which includes 64 candidate pathogenic genes for cardiomyopathies. Sanger sequencing was used to validate suspicious mutation sites and screened family members in 8 cases and 100 normal individuals,and analyzed the clinical phenotype of 4 cases of mutant carriers. The genotype-phenotype relationship in the family was analyzed. Results A frameshift mutation,c. 1377delC,was identified in the MYBPC3 gene in four alive family members,including the proband,which was not detected in 100 normal controls. Two mutation carriers were diagnosed with HCM,with the involvement of ventricular septum. The other two asymptomatic mutation carriers were found without positive finding in electrocardiogram and echocardiogram. In this pedigree,sudden cardiac death occurred in 3 family members,while syncope was presented in 4 members. Onset age less than 40 was observed in three members,while two members were diagnosed HCM after the age of 40. Conclusions Genetic test is very important in the differential diagnosis and pedigree screening of HCM.

Objective To elucidate the clinical characteristics and genotype-phenotype relationships of Chinese patients with neonatal diabetes mellitus (NDM) caused by ATP-sensitive potassium channel (KATP ) gene mutations (KATP-NDM). Methods Total of 23 suspected patients with NDM were recruited from Peking Union Medical College Hospital. Amplification and direct sequencing of all exons and exon-intron boundaries of the KCNJ11 and ABCC8 gene were done for molecular genetic diagnosis. Patients with a definite diagnosis of KATP-NDM were switched from insulin injection to oral glibenclamide. Results Thirteen patients with KATP-NDM were identified, including 9 cases of KCNJ11-NDM and 4 cases of ABCC8-NDM. Four novel mutations of KATP were identified: 1 in KCNJ11 (I182M) and 3 in ABCC8 (Q211R, I585F, R653W). Among 9 patients with KCNJ11-NDM, 2 were transient NDM (TNDM) and 7 were permanent NDM (PNDM), and 3 of which had intermediate developmental delay, neonatal diabetes (iDEND syndrome). Four patients with ABCC8-NDM were all PNDM, 2 of them had iDEND syndrome. Switching from insulin to glibenclamide monotherapy was successful in 9 patients (69.2%). Conclusions Different clinical severities and treatment responses to sulfonylureas exist even among patients with the same mutation or with different amino acids at the same mutation site. Early diagnosis of KATP-NDM and initiation of sulfonylureas are of great importance in the prevention of neurological abnormalities in patients with iDEND syndrome.

Background: A mutation in FBN1 is primarily attributed to Marfan syndrome (MFS). So far, > 1800 unique FBN1 mutations have been identified, with the vast majority being single-nucleotide substitutions, small deletions, and insertions. The rearrangement of large fragments of FBN1 accounts for only 1.7% of all variants. The aim of this study was to investigate the characteristics of large genomic rearrangements in FBN1 among MFS patients and to evaluate the correlations between genotype and phenotype. Methods: Systematic sequencing of the disease-related genes FBN1, TGFBR1, and TGFBR2, was carried out previously for 26 unrelated patients with MFS. No small mutations were found. Subsequently, multiplex ligation-dependent probe amplification was performed for the detection of copy number variations in these patients. The breakpoints were determined by gap PCR and sequencing. Transcription level analysis was conducted in patients whose RNA sample was available. Results: Four gross deletions were identified in FBN1. Three deletions (exons 6, 48-53, and 49-50) were predicted to be in-frame deletions; the remaining deletion (exons 1-36) was expected to induce the loss of one copy of the FBN1 gene. The breakpoints of these four deletions were cloned, and revealed deletion sizes of 16,551, 10,346, 4563, and 187,047 bp, respectively. Patients with in-frame deletions of exons 48-53 and 49-50 showed severe clinical phenotypes; Patient with an exon 6 deletion showed mild potential MFS phenotypes. And the patient had classic MFS with a deletion of exons 1-36. Conclusions: We characterized four large genomic rearrangements in FBN1. FBN1 haploinsufficiency correlated with a classic MFS phenotype, while in-frame deletions between exons 24-53 of FBN1 tended to cause severe clinical phenotypes.

PRKAG2 syndrome is a rare autosomal dominant inherited disorder that is characterized by cardiac hypertrophy, ventricular pre-excitation and conduction system abnormalities. There is little knowledge in cardiovascular magnetic resonance (CMR) characteristics of PRKAG2 cardiomyopathy. This study investigated the genetic defect in a three-generation Chinese family with cardiac hypertrophy and ventricular pre-excitation using whole-exome sequencing. A novel missense mutation, c.1006 G > T (p.V336L), was identified in PRKAG2 . This mutation had not been identified in the ExAC database, and the prediction result of MutationTaster indicated a deleterious effect. Furthermore, it cosegregated with the disease in the present family and was absent in unrelated 300 healthy controls. cDNA analysis did not detect any splicing defects, although the variant occurred in the first base of exon 9. CMR evaluation in five affected members showed diffuse hypertrophy in a concentric pattern, with markedly increased left ventricular mass above age and gender limits (median 151.3 g/m 2 , range 108.4–233.4 g/m 2 ). Two patients in progressive stage and one patient with sudden cardiac death exhibited extensive subendocardial late gadolinium enhancement. In conclusion, molecular screening for PRKAG2 mutations should be considered in patients who exhibit cardiac hypertrophy coexisting with ventricular pre-excitation. CMR offers promising advantages for evaluation of PRKAG2 cardiomyopathy.