Methylated miRNAs may serve as potential biomarkers and therapeutic targets for hidradenitis suppurativa.

BACKGROUND: Hidradenitis suppurativa (HS) is a chronic inflammatory disease influenced by genetics, non-genetic and environmental factors that modulate miRNA expression. Currently, no miRNA data are available for HS. In this study, we profiled DNA methylation patterns of miRNA genes associated with HS susceptibility. OBJECTIVES: Identify miRNA gene methylation profiles associated with HS susceptibility. This study examined the methylation patterns of DNAs from 24 healthy controls and 24 patients with HS using Illumina Infinium MethylationEPIC BeadChip array analysis. Methylation patterns of miRNA genes were analysed using KEGG pathway analysis to explore the inversely correlated pathways regulated by miRNAs. RESULTS: We identified 60 CpG sites representing 65 unique microRNA genes including 54 hypomethylated and 6 hypermethylated CpGs as potentially associated with HS. Some of these CpGs were found to be critical for skin function, such as miR-29, miR-200, miR-205, miR-548 and miR-132. The miR-192 is implicated in non-alcoholic fatty liver disease. The miR-200c gene was identified as a vital determinant in regulating skin repair after injury and may contribute to age-associated alterations in wound repair. miR-132 was significantly upregulated during the inflammation phase of wound repair, enhancing the activity of STAT3 and ERK pathways that promote keratinocyte proliferation. CONCLUSIONS: Epigenetically altered microRNA genes are implicated in wound healing, inflammation, keratinocyte proliferation and wound modulation. This is the first study to analyse methylation profiles of miRNA genes in the HS population, highlighting the unique role that miRNAs might play in diagnosing and treating HS.

Artificial neural networks allow response prediction in squamous cell carcinoma of the scalp treated with radiotherapy.

BACKGROUND: Epithelial neoplasms of the scalp account for approximately 2% of all skin cancers and for about 10-20% of the tumours affecting the head and neck area. Radiotherapy is suggested for localized cutaneous squamous cell carcinomas (cSCC) without lymph node involvement, multiple or extensive lesions, for patients refusing surgery, for patients with a poor general medical status, as adjuvant for incompletely excised lesions and/or as a palliative treatment. To date, prognostic risk factors in scalp cSCC patients are poorly characterized. OBJECTIVE: To identify patterns of patients with higher risk of postradiotherapy recurrence. METHODS: A retrospective observational study was performed on scalp cSCC patients with histological diagnosis who underwent conventional radiotherapy (50-120 kV) (between 1996 and 2008, follow-up from 1 to 140 months, median 14 months). Out of the 79 enrolled patients, 22 (27.8%) had previously undergone a surgery. Two months after radiotherapy, 66 (83.5%) patients achieved a complete remission, 6 (7.6%) a partial remission, whereas 2 (2.5%) proved non-responsive to the treatment and 5 cases were lost to follow-up. Demographical and clinical data were preliminarily analysed with classical descriptive statistics and with principal component analysis. All data were then re-evaluated with a machine learning-based approach using a 4th generation artificial neural networks (ANNs)-based algorithm. RESULTS: Artificial neural networks analysis revealed four scalp cSCC profiles among radiotherapy responsive patients, not previously described: namely, (i) stage T2 cSCC type, aged 70-80 years; (ii) frontal cSCC type, aged <70 years; (iii) non-recurrent nodular or nodulo-ulcerated, stage T3 cSCC type, of the vertex and treated with >60 Grays (Gy); and (iv) flat, occipital, stage T1 cSCC type, treated with 50-59 Gy. The model uncovering these four predictive profiles displayed 85.7% sensitivity, 97.6% specificity and 91.7% overall accuracy. CONCLUSIONS: Patient profiling/phenotyping with machine learning may be a new, helpful method to stratify patients with scalp cSCCs who may benefit from a RT-treatment.

Placental DNA methylation changes in detection of tetralogy of Fallot.

OBJECTIVES: To determine whether the methylation level of cytosine nucleotides in placental DNA can be used to predict tetralogy of Fallot (TOF) and provide insights into the developmental mechanism of this condition. METHODS: Tissue sections were obtained from formalin-fixed paraffin-embedded specimens of placental tissue obtained at birth from eight cases with non-chromosomal, non-syndromic TOF and 10 unaffected newborns. The Illumina Infinium HumanMethylation450 BeadChip assay was used to measure cytosine ('CpG' or 'cg') methylation levels at loci throughout the placental genome. Differential methylation was assessed by comparing the ß-values (a measure of the extent of cytosine methylation) for individual CpG loci in fetuses with TOF vs in controls. The most discriminating CpG sites were determined based on a preset cut-off of ≥ 2.0-fold change in the methylation level. The predictive accuracy of CpG loci with significant methylation changes for TOF was determined by the area under the receiver-operating-characteristics curve (AUC). A false-discovery-rate (FDR) P-value < 0.05 was used to define a statistically significant difference in the methylation level. Ingenuity Pathway Analysis (IPA) (Qiagen) was used to identify gene pathways that were significantly overexpressed, and thus altered, in TOF cases compared with controls. RESULTS: We found a total of 165 significantly differentially methylated CpG loci in TOF cases compared with controls, in 165 separate genes. These biomarkers demonstrated from fair to excellent individual predictive accuracy for TOF detection, with AUCs ≥ 0.75 (FDR P-value < 0.001 for all). The following CpG loci (gene) had the highest predictive accuracy: cg05273049 (ARHGAP22; AUC = 1.00; 95% CI, 1.00-1.00), cg02540011 (CDK5; AUC = 0.96; 95% CI, 0.87-1.00), cg08404201 (TRIM27; AUC = 0.95; 95% CI, 0.84-1.00) and cg00687252 (IER3; AUC = 0.95; 95% CI, 0.84-1.00). IPA revealed over-representation (dysregulation) of 14 gene pathways involved in normal cardiac development, including cardiomyocyte differentiation via bone morphogenetic protein receptors, cardiac hypertrophy signaling and role of nuclear factor of activated T cells in cardiac hypertrophy. Cardiac hypertrophy is an important feature of TOF. CONCLUSIONS: Analysis of placental DNA cytosine methylation changes yielded accurate markers for TOF detection and provided mechanistic information on TOF development. Our work appears to confirm the central role of epigenetic changes and of the placenta in the development of TOF. Copyright © 2019 ISUOG. Published by John Wiley & Sons Ltd.

A new species of Microhyla Tschudi, 1838 (Anura: Microhylidae) from West Coast of India: an integrative taxonomic approach.

A new species of microhylid frog Microhyla kodial sp. nov. from the west coast of India is described in this paper. It is distinct from all described species of Microhyla occurring in South and Southeast Asia as revealed by a combination of morphological, molecular and acoustic characters. The new species is characterized by absence of lateral body stripe, tuberculated dorsal skin surface, absence of webbing between fingers, presence of basal webbing between toes and absence of dorsal marginal groove on finger and toe disc. Each male advertisement call lasts for 0.11-0.42 s and is comprised of 2-7 pulses with a dominant frequency of 3.3-4.2 kHz. The breeding season is short, limited to the rainy season (June to September) and the females lay up to 300 eggs per clutch. A molecular phylogenetic tree constructed using the mitochondrial 16S rRNA gene shows that M. kodial sp. nov. is closely related to the M. achatina group from Southeast Asia. The uncorrected genetic divergence between the new species and its closest congeners M. heymonsi, M. mantheyi, M. borneensis and M. orientalis were 7.3-7.6 %, 7.5-7.8%, 7.8-8.1% and 8.1-8.4% respectively. At present, this species is known only from the type locality, a highly disturbed urban and industrialized area which needs conservation intervention.

Haplotypes, mutations and male fertility: the story of the testis-specific ubiquitin protease USP26.

Recently, mutations in the X-linked ubiquitin protease 26 (USP26) gene have been proposed to be associated with male infertility. In particular a 371insACA, 494T>C and 1423C>T haplotype, which results in a T123-124ins, L165S and H475Y amino acid change respectively, has been reported to be associated with Sertoli cell-only syndrome (SCOS) and an absence of sperm in the ejaculate. Here, we demonstrate that two of these changes actually correspond to the ancestral sequence of the gene and that the USP26 haplotype is present in significant frequencies in sub-Saharan African and South and East Asian populations, including in individuals with known fertility. This indicates that the allele is not associated with infertility. The pattern of frequency distribution of the derived haplotype (371delACA, 494T), which is present at high frequencies in most non-African populations could be interpreted as either a result of migration followed by simple genetic drift or alternatively as positive selection acting on the derived alleles. The latter hypothesis seems likely, because there is evidence of strong positive selection acting on the USP26 gene.

Search for the second Peutz-Jeghers syndrome locus: exclusion of the STK13, PRKCG, KLK10, and PSCD2 genes on chromosome 19 and the STK11IP gene on chromosome 2.

Pathogenic mutations in the serine/threonine kinase STK11 (alias LKB1) cause Peutz-Jeghers syndrome (PJS) in most affected individuals. However, in a considerable number of PJS-patients mutations cannot be detected in STK11 suggesting genetic heterogeneity. One PJS family without STK11 mutations (PJS07) has previously been described with significant evidence for linkage to a second potential PJS locus on 19q13.3-->q13.4. In this study we investigated candidate genes within markers D19S180 and D19S254, since multipoint linkage analysis yielded significant LOD scores for this region in this family. Four genes in the region (cytohesin 2: PSCD2, kallikrein 10: KLK10, protein kinase C gamma: PRKCG, and serine/threonine kinase 13: STK13) potentially involved in growth inhibitory pathways or in the pathophysiology of can- cer, were considered as candidates. We first determined the genomic structure of the PSCD2 and PRKCG genes, and performed mutation analysis of all exons and exon-intron junctions of the four genes, in the PJS07 family. No pathogenic mutation was identified in these four genes in affected individuals. A very rare polymorphism resulting in a conserved amino acid change Lys to Arg was found in PSCD2. These data provide considerable evidence for exclusion of these four genes as candidates for the second locus on 19q13.3-->q13.4 in PJS. Finally, we also excluded the recently identified STK11-interacting protein gene (STK11IP, alias LIP1) mapped in 2q36 as candidate for PJS in the PJS07 family, although this could be a good candidate in other non-STK11/LKB1 families.

An apparently dominant bipolar affective disorder (BPAD) locus on chromosome 20p11.2-q11.2 in a large Turkish pedigree.

Bipolar affective disorder (BPAD), also known as manic-depressive illness, is a common complex, polygenic disorder characterised by recurrent cyclic episodes of mania and depression. Family, twin, and adoption studies strongly suggest a genetic predisposition/susceptibility to BPAD, but no genes have yet been identified. We studied a large Turkish pedigree, with an apparently autosomal dominant BPAD, which contained 13 affected individuals. The age of onset ranged from 15-40 with a mean of 25 years. The phenotypes consisted of recurrent manic and major depressive episodes, including suicidal attempts; there was usually full remission with lithium treatment. A genome-wide linkage analysis using a dominant mode of inheritance showed strong evidence for a BPAD susceptibility locus on chromosome 20p11.2-q11.2. The highest 2-point lod score of 4.34 at theta = 0 was obtained with markers D20S604, D20S470, D20S836 and D20S838 using a dominant model with full penetrance. Haplotype analysis enabled the mapping of the BPAD locus in this family between markers D20S186 and D20S109, to a region of approximately 42 cM.

Insertion of beta-satellite repeats identifies a transmembrane protease causing both congenital and childhood onset autosomal recessive deafness.

Approximately 50% of childhood deafness is caused by mutations in specific genes. Autosomal recessive loci account for approximately 80% of nonsyndromic genetic deafness. Here we report the identification of a new transmembrane serine protease (TMPRSS3; also known as ECHOS1) expressed in many tissues, including fetal cochlea, which is mutated in the families used to describe both the DFNB10 and DFNB8 loci. An 8-bp deletion and insertion of 18 monomeric (approximately 68-bp) beta-satellite repeat units, normally present in tandem arrays of up to several hundred kilobases on the short arms of acrocentric chromosomes, causes congenital deafness (DFNB10). A mutation in a splice-acceptor site, resulting in a 4-bp insertion in the mRNA and a frameshift, was detected in childhood onset deafness (DFNB8). This is the first description of beta-satellite insertion into an active gene resulting in a pathogenic state, and the first description of a protease involved in hearing loss.

Y-chromosome lineages trace diffusion of people and languages in southwestern Asia.

The origins and dispersal of farming and pastoral nomadism in southwestern Asia are complex, and there is controversy about whether they were associated with cultural transmission or demic diffusion. In addition, the spread of these technological innovations has been associated with the dispersal of Dravidian and Indo-Iranian languages in southwestern Asia. Here we present genetic evidence for the occurrence of two major population movements, supporting a model of demic diffusion of early farmers from southwestern Iran-and of pastoral nomads from western and central Asia-into India, associated with Dravidian and Indo-European-language dispersals, respectively.

Clouston hidrotic ectodermal dysplasia (HED): genetic homogeneity, presence of a founder effect in the French Canadian population and fine genetic mapping.

HED is an autosomal dominant skin disorder that is particularly common in the French Canadian population of south-west Quebec. We previously mapped the HED gene to the pericentromeric region of chromosome 13q using linkage analysis in eight French Canadian families. In this study, we extend our genetic analysis to include a multiethnic group of 29 families with 10 polymorphic markers spanning 5.1 cM in the candidate region. Two-point linkage analysis strongly suggests absence of genetic heterogeneity in HED in four families of French, Spanish, African and Malaysian origins. Multipoint linkage analysis in all 29 families generated a peak lod score of 53.5 at D13S1835 with a 1 lod unit support interval spanning 1.8 cM. Recombination mapping placed the HED gene in a 2.4 cM region flanked by D13S1828 proximally and D13S1830 distally. We next show evidence for a strong founder effect in families of French Canadian origin thereby representing the first example of a founder disease in the south-west part of the province of Quebec. Significant association was found between HED in these families and all markers analysed (Fisher's exact test, P < 0.001). Complete allelic association was detected at D13S1828, D13S1827, D13S1835, D13S141 and D13S175 (P(excess) = 1) spanning 1.3 cM. A major haplotype including all 10 associated alleles was present on 65% of affected chromosomes. This haplotype most likely represents the founder haplotype that introduced the HED mutation into the French Canadian population. Luria-Delbrück equations and multipoint likelihood linkage disequilibrium analysis positioned the gene at the D13S1828 locus (likely range estimate: 1.75 cM) and 0.58 cM telomeric to this marker (support interval: 3.27 cM) respectively.

Primary ciliary dyskinesia: a genome-wide linkage analysis reveals extensive locus heterogeneity.

Primary ciliary dyskinesia (PCD), or immotile cilia syndrome (ICS), is an autosomal recessive disorder affecting ciliary movement with an incidence of 1 in 20000-30000. Dysmotility to complete immotility of cilia results in a multisystem disease of variable severity with recurrent respiratory tract infections leading to bronchiectasis and male subfertility. Ultrastructural defects are present in ciliated mucosa and spermatozoa. Situs inversus (SI) is found in about half of the patients (Kartagener syndrome). We have collected samples from 61 European and North American families with PCD. A genome-wide linkage search was performed in 31 multiplex families (169 individuals including 70 affecteds) using 188 evenly spaced (19cM average interval) polymorphic markers. Both parametric (recessive model) and non-parametric (identity by descent allele sharing) linkage analyses were used. No major locus for the majority of the families was identified, although the sample was powerful enough to detect linkage if 40% of the families were linked to one locus. These results strongly suggest extensive locus heterogeneity. Potential genomic regions harbouring PCD loci were localised on chromosomes 3p, 4q, 5p, 7p, 8q, 10p, 11q, 13q, 15q, 16p, 17q and 19q. Linkage analysis using PCD families with a dynein arm deficiency provided 'suggestive' evidence for linkage to chromosomal regions 8q, 16pter, while analyses using only PCD families with situs inversus resulted in 'suggestive' scores for chromosomes 8q, and 19q.

The phenotypic spectrum of GLI3 morphopathies includes autosomal dominant preaxial polydactyly type-IV and postaxial polydactyly type-A/B; No phenotype prediction from the position of GLI3 mutations.

Functional characterization of a gene often requires the discovery of the full spectrum of its associated phenotypes. Mutations in the human GLI3 gene have been identified in Greig cepalopolysyndactyly, Pallister-Hall syndrome (PHS), and postaxial polydactyly type-A (PAP-A). We studied the involvement of GLI3 in additional phenotypes of digital abnormalities in one family (UR003) with preaxial polydactyly type-IV (PPD-IV), three families (UR014, UR015, and UR016) with dominant PAP-A/B (with PPD-A and -B in the same family), and one family with PHS. Linkage analysis showed no recombination with GLI3-linked polymorphisms. Family UR003 had a 1-nt frameshift insertion, resulting in a truncated protein of 1,245 amino acids. A frameshift mutation due to a 1-nt deletion was found in family UR014, resulting in a truncated protein of 1,280 amino acids. Family UR015 had a nonsense mutation, R643X, and family UR016 had a missense mutation, G727R, in a highly conserved amino acid of domain 3. The patient with PHS had a nonsense mutation, E1147X. These results add two phenotypes to the phenotypic spectrum caused by GLI3 mutations: the combined PAP-A/B and PPD-IV. These mutations do not support the suggested association between the mutations in GLI3 and the resulting phenotypes. We propose that all phenotypes associated with GLI3 mutations be called "GLI3 morphopathies," since the phenotypic borders of the resulting syndromes are not well defined and there is no apparent genotype-phenotype correlation.

Lack of linkage or association between schizophrenia and the polymorphic trinucleotide repeat within the KCNN3 gene on chromosome 1q21.

To determine the importance of a candidate gene KCNN3 (formerly named hSKCa3) in the susceptibility to schizophrenia, we have studied the genotypes of a (CAG)n polymorphism within this gene in the DNAs of the members of 54 multiplex families with this disease. Parametric and nonparametric linkage analysis did not provide evidence for linkage between KCNN3 (that we mapped to chromosome 1q21) and schizophrenia. Furthermore, we observed no difference in the distribution of the (CAG)n alleles between affected and normal individuals. These results do not support the hypothesis that larger KCNN3 alleles are preferentially associated with schizophrenia [Chandy et al. 1998 Mol Psychiatr 3:32-37] in individuals from multiply affected families.

No evidence for linkage between schizophrenia and markers at chromosome 15q13-14.

Freedman et al. [1997: Proc Natl Acad Sci USA 94:587-592] reported linkage in nine multiplex schizophrenia families to markers on chromosome 15, using impaired neuronal inhibition to repeated auditory stimuli (P50), a neurophysiological deficit associated with schizophrenia, as the phenotype. The highest LOD score obtained (5.3 at theta = 0) was for marker D15S1360 mapped to chromosome 15q13-14, less than 120 kb from the alpha7-nicotinic receptor (CHRNA7) gene. The study also reported a small positive LOD score for D15S1360 when examined for linkage to the schizophrenia phenotype. Following these findings, we examined three polymorphic markers (D15S1360, L76630, and ACTC) on chromosome 15q13-14 near the CHRNA7 gene for linkage to schizophrenia, using 54 pedigrees from an independent study. Alleles for these three markers were genotyped and analyzed using parametric and nonparametric methods. No LOD score above 1.00 was obtained for any marker, and affected sib-pair analysis likewise showed no evidence for linkage. We conclude that in our families the region around the CHRNA7 locus does not contain a major locus for susceptibility to schizophrenia.

Schizophrenia susceptibility loci on chromosomes 13q32 and 8p21.

Schizophrenia is a common disorder characterized by psychotic symptoms; diagnostic criteria have been established. Family, twin and adoption studies suggest that both genetic and environmental factors influence susceptibility (heritability is approximately 71%; ref. 2), however, little is known about the aetiology of schizophrenia. Clinical and family studies suggest aetiological heterogeneity. Previously, we reported that regions on chromosomes 22, 3 and 8 may be associated with susceptibility to schizophrenia, and collaborations provided some support for regions on chromosomes 8 and 22 (refs 9-13). We present here a genome-wide scan for schizophrenia susceptibility loci (SSL) using 452 microsatellite markers on 54 multiplex pedigrees. Non-parametric linkage (NPL) analysis provided significant evidence for an SSL on chromosome 13q32 (NPL score=4.18; P=0.00002), and suggestive evidence for another SSL on chromosome 8p21-22 (NPL=3.64; P=0.0001). Parametric linkage analysis provided additional support for these SSL. Linkage evidence at chromosome 8 is weaker than that at chromosome 13, so it is more probable that chromosome 8 may be a false positive linkage. Additional putative SSL were noted on chromosomes 14q13 (NPL=2.57; P=0.005), 7q11 (NPL=2.50, P=0.007) and 22q11 (NPL=2.42, P=0.009). Verification of suggestive SSL on chromosomes 13q and 8p was attempted in a follow-up sample of 51 multiplex pedigrees. This analysis confirmed the SSL in 13q14-q33 (NPL=2.36, P=0.007) and supported the SSL in 8p22-p21 (NPL=1.95, P=0.023).

Peutz-Jeghers syndrome: confirmation of linkage to chromosome 19p13.3 and identification of a potential second locus, on 19q13.4.

Peutz-Jeghers syndrome (PJS) is an autosomal dominant disease with variable expression and incomplete penetrance, characterized by mucocutaneous pigmentation and hamartomatous polyposis. Patients with PJS have increased frequency of gastrointestinal and extraintestinal malignancies (ovaries, testes, and breast). In order to map the locus (or loci) associated with PJS, we performed a genomewide linkage analysis, using DNA polymorphisms in six families (two from Spain, two from India, one from the United States, and one from Portugal) comprising a total of 93 individuals, including 39 affected and 48 unaffected individuals and 6 individuals with unknown status. During this study, localization of a PJS gene to 19p13.3 (around marker D19S886) had been reported elsewhere. For our families, marker D19S886 yielded a maximum LOD score of 4.74 at a recombination fraction (theta) of .045; multipoint linkage analysis resulted in a LOD score of 7.51 for the interval between D19S886 and 19 pter. However, markers on 19q13.4 also showed significant evidence for linkage. For example, D19S880 resulted in a maximum LOD score of 3.8 at theta = .13. Most of this positive linkage was contributed by a single family, PJS07. These results confirm the mapping of a common PJS locus on 19p13.3 but also suggest the existence, in a minority of families, of a potential second PJS locus, on 19q13.4. Positional cloning and characterization of the PJS mutations will clarify the genetics of the syndrome and the implication of the gene(s) in the predisposition to neoplasias.