Bi-Allelic Pathogenic Variations in MERTK Including Deletions Are Associated with an Early Onset Progressive Form of Retinitis Pigmentosa.

Bi-allelic pathogenic variants in MERTK cause retinitis pigmentosa (RP). Since deletions of more than one exon have been reported repeatedly for MERTK, CNV (copy number variation) analysis of next-generation sequencing (NGS) data has proven important in molecular genetic diagnostics of MERTK. CNV analysis was performed on NGS data of 677 individuals with inherited retinal diseases (IRD) and confirmed by quantitative RT-PCR analysis. Clinical evaluation was based on retrospective records. Clinical re-examination included visual field examination, dark adaption, scotopic and photopic full-field electroretinograms (ffERG), multifocal ERG (mfERG) and optic coherence tomography (OCT). Fourteen variants were detected in MERTK in six individuals, three of which were deletions of more than one exon. Clinical examinations of five out of six individuals revealed a severe phenotype with early-onset generalized retinal dystrophy with night blindness and progressive visual field loss; however, one individual had a milder phenotype. Three individuals had hearing impairments. We show that deletions represent a substantial part of the causative variants in MERTK and emphasize that CNV analysis should be included in the molecular genetic diagnostics of IRDs.

A Missense Mutation in RAB28 in a Family with Cone-Rod Dystrophy and Postaxial Polydactyly Prevents Localization of RAB28 to the Primary Cilium.

Purpose: Cone-rod dystrophy (CRD) is a rare hereditary eye disorder that causes progressive degeneration of cone and rod photoreceptors. More than 30 genes, including RAB28, have been associated with CRD; however, only a few RAB28 variants have been reported to be associated with CRD. In this study, we describe two brothers with CRD and a homozygous missense variant, c.55G>A (p.Gly19Arg), in RAB28. Methods: The missense variant was identified as part of a study investigating underlying genetic defects in a large patient cohort (n = 667) using targeted next-generation sequencing of 125 genes associated with retinal dystrophy. Cellular localization of RAB28 and ciliogenesis in patient fibroblasts were investigated by immunofluorescence microscopy. The effect of the missense variant on RAB28 expression level was investigated by quantitative real-time PCR. Results: Two brothers of a consanguineous couple presented with CRD, postaxial polydactyly (PAP), and myopia. Both brothers had a homozygous missense RAB28 variant located in the G1 box of the guanosine triphosphate/guanosine diphosphate binding domain of RAB28. This missense variant caused a considerable reduction of RAB28 localized to the cilia, whereas ciliogenesis seemed unaffected. Conclusions: The missense variant in RAB28 is classified as likely pathogenic with functional effect on protein localization. The combination of retinal dystrophy and PAP are well known from ciliopathies; however, more data are needed to finally conclude that the RAB28 variant described here is the cause of PAP in these brothers.

Molecular genetic analysis using targeted NGS analysis of 677 individuals with retinal dystrophy.

Inherited retinal diseases (IRDs) are a common cause of visual impairment. IRD covers a set of genetically highly heterogeneous disorders with more than 150 genes associated with one or more clinical forms of IRD. Molecular genetic diagnosis has become increasingly important especially due to expanding number of gene therapy strategies under development. Next generation sequencing (NGS) of gene panels has proven a valuable diagnostic tool in IRD. We present the molecular findings of 677 individuals, residing in Denmark, with IRD and report 806 variants of which 187 are novel. We found that deletions and duplications spanning one or more exons can explain 3% of the cases, and thus copy number variation (CNV) analysis is important in molecular genetic diagnostics of IRD. Seven percent of the individuals have variants classified as pathogenic or likely-pathogenic in more than one gene. Possible Danish founder variants in EYS and RP1 are reported. A significant number of variants were classified as variants with unknown significance; reporting of these will hopefully contribute to the elucidation of the actual clinical consequence making the classification less troublesome in the future. In conclusion, this study underlines the relevance of performing targeted sequencing of IRD including CNV analysis as well as the importance of interaction with clinical diagnoses.

A pathogenic haplotype, common in Europeans, causes autosomal recessive albinism and uncovers missing heritability in OCA1.

Oculocutaneous albinism (OCA) is a genetically heterogeneous disorder. Six genes are associated with autosomal recessive OCA (TYR, OCA2, TYRP1, SLC45A2, SLC24A5 and LRMDA), and one gene, GPR143, is associated with X-linked ocular albinism (OA). Molecular genetic analysis provides a genetic diagnosis in approximately 60% of individuals with clinical OA/OCA. A considerably number of the remaining 40% are heterozygous for a causative sequence variation in TYR. To identify missing causative sequence variants in these, we used a NGS based approach, genotyping and segregation analysis. We report two putative pathogenic haplotypes which only differ by two extremely rare SNVs, indicating that the haplotypes have a common derivation. Both haplotypes segregate consistent with an autosomal recessive inheritance pattern and include the allele p.S192Y-p.R402Q. An explanation for the pathogenicity of the haplotypes could be the combination of p.S192Y and p.R402Q. Homozygosity for the pathogenic haplotypes causes a partial albinism phenotype. In our cohort, 15% of affected individuals had a molecular genetic diagnosis involving the pathogenic haplotype. Consequently, the prevalence of albinism seems to be substantially underestimated, and children with unexplained bilateral subnormal vision and/or nystagmus should be analysed clinically and molecularly for albinism.

Mosaic MECP2 variants in males with classical Rett syndrome features, including stereotypical hand movements.

Rett syndrome is rarely suspected in males because of the X-linked dominant inheritance. In the literature, only six male patients have been reported with methyl-CpG-binding protein 2 (MECP2) mosaicism. Next-generation sequencing (NGS) methods have enabled better detection of somatic mosaicism compared to conventional Sanger sequencing; however, mosaics can still be difficult to detect. We present clinical and molecular findings in two males mosaic for a pathogenic MECP2 variant. Both have been reexamined using deep sequencing of DNA isolated from four different cell tissues (blood, muscle, fibroblasts and oral mucosa). Deep sequencing of the different tissues revealed that the variants were present in all tissues. In one patient, the molecular diagnosis could only be established by reexamination after a normal whole exome sequencing, and the other case is an example of reverse genetic diagnostics. Rett syndrome should be considered in males with neurodevelopmental delay and stereotypical hand movements. Subsequent to clinical diagnosis males should be investigated with NGS-based technologies of MECP2 with high read depth and a low threshold for variant calls. If the initial analysis on full blood derived DNA fails to confirm the suspicion, we recommend repeating the analysis on another tissue, preferentially fibroblasts to increase the diagnostic yield.

Replicative and non-replicative mechanisms in the formation of clustered CNVs are indicated by whole genome characterization.

Clustered copy number variants (CNVs) as detected by chromosomal microarray analysis (CMA) are often reported as germline chromothripsis. However, such cases might need further investigations by massive parallel whole genome sequencing (WGS) in order to accurately define the underlying complex rearrangement, predict the occurrence mechanisms and identify additional complexities. Here, we utilized WGS to delineate the rearrangement structure of 21 clustered CNV carriers first investigated by CMA and identified a total of 83 breakpoint junctions (BPJs). The rearrangements were further sub-classified depending on the patterns observed: I) Cases with only deletions (n = 8) often had additional structural rearrangements, such as insertions and inversions typical to chromothripsis; II) cases with only duplications (n = 7) or III) combinations of deletions and duplications (n = 6) demonstrated mostly interspersed duplications and BPJs enriched with microhomology. In two cases the rearrangement mutational signatures indicated both a breakage-fusion-bridge cycle process and haltered formation of a ring chromosome. Finally, we observed two cases with Alu- and LINE-mediated rearrangements as well as two unrelated individuals with seemingly identical clustered CNVs on 2p25.3, possibly a rare European founder rearrangement. In conclusion, through detailed characterization of the derivative chromosomes we show that multiple mechanisms are likely involved in the formation of clustered CNVs and add further evidence for chromoanagenesis mechanisms in both "simple" and highly complex chromosomal rearrangements. Finally, WGS characterization adds positional information, important for a correct clinical interpretation and deciphering mechanisms involved in the formation of these rearrangements.

Heterozygous mutations in GTP-cyclohydrolase-1 reduce BH4 biosynthesis but not pain sensitivity.

Human studies have demonstrated a correlation between noncoding polymorphisms of "the pain protective" haplotype in the GCH1 gene that encodes for GTP cyclohydrolase I (GTPCH1)-which leads to reduced tetrahydrobiopterin (BH4) production in cell systems-and a diminished perception of experimental and clinical pain. Here, we investigate whether heterozygous mutations in the GCH1 gene which lead to a profound BH4 reduction in patients with dopa-responsive dystonia (DRD) have any effect on pain sensitivity. The study includes an investigation of GCH1-associated biomarkers and pain sensitivity in a cohort of 22 patients with DRD and 36 controls. The patients with DRD had, when compared with controls, significantly reduced levels of BH4, neopterin, biopterin, and GTPCH1 in their urine, blood, or cytokine-stimulated fibroblasts, but their pain response with respect to non-painful stimulation, (acute) stimulus-evoked pain, or pain response after capsaicin-induced sensitization was not significantly different. A family-specific cohort of 11 patients with DRD and 11 controls were included in this study. The patients with DRD were heterozygous for the pain protective haplotype in cis with the GCH1 disease-causing mutation, c.899T>C. No effect on pain perception was observed for this combined haplotype. In conclusion, a reduced concentration of BH4 is not sufficient to alter ongoing pain sensitivity or evoked pain responses.

Phenotypes and genotypes in individuals with SMC1A variants.

SMC1A encodes one of the proteins of the cohesin complex. SMC1A variants are known to cause a phenotype resembling Cornelia de Lange syndrome (CdLS). Exome sequencing has allowed recognizing SMC1A variants in individuals with encephalopathy with epilepsy who do not resemble CdLS. We performed an international, interdisciplinary study on 51 individuals with SMC1A variants for physical and behavioral characteristics, and compare results to those in 67 individuals with NIPBL variants. For the Netherlands all known individuals with SMC1A variants were studied, both with and without CdLS phenotype. Individuals with SMC1A variants can resemble CdLS, but manifestations are less marked compared to individuals with NIPBL variants: growth is less disturbed, facial signs are less marked (except for periocular signs and thin upper vermillion), there are no major limb anomalies, and they have a higher level of cognitive and adaptive functioning. Self-injurious behavior is more frequent and more severe in the NIPBL group. In the Dutch group 5 of 13 individuals (all females) had a phenotype that shows a remarkable resemblance to Rett syndrome: epileptic encephalopathy, severe or profound intellectual disability, stereotypic movements, and (in some) regression. Their missense, nonsense, and frameshift mutations are evenly spread over the gene. We conclude that SMC1A variants can result in a phenotype resembling CdLS and a phenotype resembling Rett syndrome. Resemblances between the SMC1A group and the NIPBL group suggest that a disturbed cohesin function contributes to the phenotype, but differences between these groups may also be explained by other underlying mechanisms such as moonlighting of the cohesin genes.

TS-EUROTRAIN: A European-Wide Investigation and Training Network on the Etiology and Pathophysiology of Gilles de la Tourette Syndrome.

Gilles de la Tourette Syndrome (GTS) is characterized by the presence of multiple motor and phonic tics with a fluctuating course of intensity, frequency, and severity. Up to 90% of patients with GTS present with comorbid conditions, most commonly attention-deficit/hyperactivity disorder (ADHD), and obsessive-compulsive disorder (OCD), thus providing an excellent model for the exploration of shared etiology across disorders. TS-EUROTRAIN (FP7-PEOPLE-2012-ITN, Grant Agr.No. 316978) is a Marie Curie Initial Training Network (http://ts-eurotrain.eu) that aims to elucidate the complex etiology of the onset and clinical course of GTS, investigate the neurobiological underpinnings of GTS and related disorders, translate research findings into clinical applications, and establish a pan-European infrastructure for the study of GTS. This includes the challenges of (i) assembling a large genetic database for the evaluation of the genetic architecture with high statistical power; (ii) exploring the role of gene-environment interactions including the effects of epigenetic phenomena; (iii) employing endophenotype-based approaches to understand the shared etiology between GTS, OCD, and ADHD; (iv) establishing a developmental animal model for GTS; (v) gaining new insights into the neurobiological mechanisms of GTS via cross-sectional and longitudinal neuroimaging studies; and (vi) partaking in outreach activities including the dissemination of scientific knowledge about GTS to the public. Fifteen partners from academia and industry and 12 PhD candidates pursue the project. Here, we aim to share the design of an interdisciplinary project, showcasing the potential of large-scale collaborative efforts in the field of GTS. Our ultimate aims are to elucidate the complex etiology and neurobiological underpinnings of GTS, translate research findings into clinical applications, and establish Pan-European infrastructure for the study of GTS and associated disorders.

Proximal 21q deletion as a result of a de novo unbalanced t(12;21) translocation in a patient with dysmorphic features, hepatomegaly, thick myocardium and delayed psychomotor development.

BACKGROUND: IInterstitial 21q deletions can cause a wide spectrum of symptoms depending on the size and the location of the deletion. It has previously been suggested that the long arm of chromosome 21 can be divided into three regions based on the clinical severity of the patients and deletion of the region from 32.3 Mb to 37.1 Mb was more crucial than the deletion of other regions. CASE PRESENTATION: In this study we describe a female patient with dysmorphic features, hepatomegaly, thick myocardium and psychomotor delay. Conventional karyotyping was initially interpreted as full monosomy 21, but subsequent chromosome microarray analysis suggested an approximately 18 Mb partial monosomy. Re-evaluation of the karyotype and fluorescence in situ hybridization revealed deletion of the proximal 21q11.2-q22.11 segment and insertion of 21q22.11-qter to 12qter. The deletion of the present case overlaps with two of the proposed regions including part of the proposed crucial region. CONCLUSIONS: This report emphasizes the relevance of investigating suspected full monosomies with high resolution methods and FISH in order to investigate the extent of the deletion and the presence of more complex rearrangements.

Deletion of 11q12.3-11q13.1 in a patient with intellectual disability and childhood facial features resembling Cornelia de Lange syndrome.

Deletions within 11q12.3-11q13.1 are very rare and to date only two cases have been described in the literature. In this study we describe a 23-year-old male patient with intellectual disability, behavioral problems, dysmorphic features, dysphagia, gastroesophageal reflux and skeletal abnormalities. Cornelia de Lange syndrome (CdLS, OMIM #122470; #300590; #610759; #300882; #614701) was suggested as a differential diagnosis in childhood although he lacked some of the features typical for this disorder. He does not have a mutation in any of the five known CdLS genes (NIPBL, SMC1A, SMC3, HDAC8, RAD21), but a 1.6Mb deletion at chromosome region 11q12.3-11q13.1 was detected by chromosome microarray. The deletion contains several genes including PPP2R5B, which has been associated with intellectual disability and overgrowth; NRXN2, which has been associated with intellectual disability and autism spectrum disorder; and CDCA5, which is part of the cohesin pathway, as are all the five known CdLS genes. It is therefore possible that deletion of CDCA5 may account for some of the CdLS like features of the present case.

Long QT syndrome in South Africa: the results of comprehensive genetic screening.

Congenital long QT syndrome (cLQTS) is a genetic disorder predisposing to ventricular arrhythmia, syncope and sudden death. Over 700 different cLQTS-causing mutations in 13 genes are known. The genetic spectrum of LQTS in 44 South African cLQTS patients (23 known to carry the South African founder mutation p.A341V in KCNQ1) was established by screening for mutations in the coding regions of KCNQ1, KCNH2, KCNE1, KCNE2 and SCN5A, the most frequently implicated cLQTS-causing genes (five-gene screening). Fourteen disease-causing mutations were identified, eight (including the founder mutation) in KCNQ1, five in KCNH2 and one in KCNE1. Two mutations were novel. Two double heterozygotes were found among the 23 families (8.5%) carrying the founder mutation. In conclusion, cLQTS in South Africa reflects both a strong founder effect and a genetic spectrum similar to that seen in other populations. Consequently, five-gene screening should be offered as a standard screening option, as is the case internationally. This will disclose compound and double heterozygotes. Fivegene screening will most likely be even more informative in other South African sub-populations with a greater genetic diversity.

Ring chromosome 9 in a girl with developmental delay and dysmorphic features: case report and review of the literature.

In this report, we describe a female child with dysmorphic features and developmental delay. Chromosome microarray analysis followed by conventional karyotyping revealed a ring chromosome 9 with a 12 Mb deletion at 9pter-p23 and a 540 kb deletion at 9q34.3-qter. Four percent of the analyzed cells had monosomy 9. The patient has the features of both the Kleefstra syndrome and the chromosome 9p-syndrome, including trigonocephaly, long philtrum, hypertelorism, and retro-/micronagthia. The deletion of the patient overlaps with several of the proposed critical regions for the 9p deletion syndrome.

MT-CYB mutations in hypertrophic cardiomyopathy.

Mitochondrial dysfunction is a characteristic of heart failure. Mutations in mitochondrial DNA, particularly in MT-CYB coding for cytochrome B in complex III (CIII), have been associated with isolated hypertrophic cardiomyopathy (HCM). We hypothesized that MT-CYB mutations might play an important causal or modifying role in HCM. The MT-CYB gene was sequenced from DNA isolated from blood from 91 Danish HCM probands. Nonsynonymous variants were analyzed by bioinformatics, molecular modeling and simulation. Two germline-inherited, putative disease-causing, nonsynonymous variants: m.15024G>A; p.C93Y and m.15482T>C; p.S246P were identified. Modeling showed that the p.C93Y mutation leads to disruption of the tertiary structure of Cytb by helix displacement, interfering with protein-heme interaction. The p.S246P mutation induces a diproline structure, which alters local secondary structure and induces a kink in the protein backbone, interfering with macromolecular interactions. These molecular effects are compatible with a leaky phenotype, that is, limited but progressive mitochondrial dysfunction. In conclusion, we find that rare, putative leaky mtDNA variants in MT-CYB can be identified in a cohort of HCM patients. We propose that further patients with HCM should be examined for mutations in MT-CYB in order to clarify the role of these variants.

Real-time TaqMan polymerase chain reaction-based genus-identification and pyrosequencing-based species identification of Campylobacter jejuni, C. coli, C. lari, C. upsaliensis, and C. fetus directly on stool samples.

A new method was developed for Campylobacter identification and applied directly on 599 stool samples from diarrhoeagenic patients. Here, the gyrase B gene of Campylobacter was targeted in a 2-step process: first, TaqMan polymerase chain reaction (PCR)-based identification of C. jejuni, C. coli, C. upsaliensis, C. lari, and C. fetus at the genus level, and, second, pyrosequencing-based identification at the species level. The TaqMan PCR method was compared to culturing and identified 87 Campylobacter-positive samples of which 64 were culture positive. Among the discrepant 23 samples, 18 were confirmed positive by conventional PCR, underlining a significant increase in diagnostic yield by use of this molecular and culture-independent method. For species identification, the pyrosequencing method was compared to conventional PCR and among the 87 TaqMan PCR-positive samples, 74 Campylobacter species were identified by both methods, 10 samples gave discrepant results, and 3 samples were negative by both methods.

There is no association between the circadian clock gene HPER3 and cognitive dysfunction after noncardiac surgery.

BACKGROUND: The specific clock-gene PERIOD3 is important with regard to circadian rhythmicity, sleep homeostasis, and cognitive function. The allele PER3(5/5) has been associated with worse cognitive performance in response to sleep deprivation. We hypothesized that patients with the PER3(5/5) genotype would have an increased risk of postoperative cognitive dysfunction (POCD) 1 week after noncardiac surgery. METHODS: Blood samples were analyzed from 93 patients with POCD and 186 patients without POCD from a completed multicenter study. The study population comprised patients ages 40 years and older undergoing noncardiac surgery who were tested preoperatively and 1 week after surgery with a neuropsychological test battery comprising 7 subtests. PER3 genotypes were determined by polymerase chain reaction analysis of DNA from blood samples (Clinicaltrials.gov identifier NCT01088100). RESULTS: The frequencies of the 3 genotypes were 11.8% (32 patients) PER3(5/5), 41.7% (113 patients) PER3(4/5), and 46.5% (126 patients) PER3(4/4). No significant difference was found in the distribution of the 3 genotypes according to POCD at 1 week (P = 0.68). Twelve percent (6% to 21%) of the patients with POCD and 12% (7% to 17%) of the patients without POCD had the PER3(5/5) genotype. The difference of the incidence of POCD/-POCD for the PER3(5/5) genotype was 1% (-7% to 10%). A significantly higher Z score was found in patients having the PER3(4/4) in 1 of the neuropsychological tests (error score of the Concept Shifting Test) (Bonferroni corrected P = 0.042). CONCLUSION: No significant association was found between the clock-gene PER3(5/5) genotype and POCD at 1 week after noncardiac surgery. If PER3(5/5) does worsen cognitive performance, the incidence is <10% of patients.

Genome-wide peripheral blood leukocyte DNA methylation microarrays identified a single association with inflammatory bowel diseases.

BACKGROUND: Crohn's disease (CD) and ulcerative colitis (UC) are common forms of inflammatory bowel disease (IBD). Monozygotic (MZ) twin discordance rates and epidemiologic data implicate that environmental changes and epigenetic factors may play a pathogenic role in IBD. DNA methylation (the methylation of cytosines within CpG dinucleotides) is an epigenetic modification, which can respond to environmental influences. We investigated whether DNA methylation might be connected with IBD in peripheral blood leukocyte (PBL) DNA by utilizing genome-wide microarrays. METHODS: Two different high-throughput microarray-based methods for genome-wide DNA methylation analysis were employed. First, DNA isolated from MZ twin pairs concordant (CD: 4; UC: 3) and discordant (CD: 4; UC: 7) for IBD was interrogated by a custom-made methylation-specific amplification microarray (MSAM). Second, the recently developed Illumina Infinium HumanMethylation450 BeadChip arrays were used on 48 samples of PBL DNA from discordant MZ twin pairs (CD: 3; UC: 3) and treatment-naive pediatric cases of IBD (CD: 14; UC: 8), as well as controls (n = 14). The microarrays were validated with bisulfite pyrosequencing. RESULTS: The MSAMs did not yield significant IBD associations. The Methylation BeadChip approach identified a single DNA methylation association of IBD at TEPP (testis, prostate and placenta-expressed protein) when DNA isolated selectively from peripheral blood mononuclear cells was analyzed (8.6% increase in methylation between CD and control, FDR = 0.0065). CONCLUSIONS: Microarray interrogation of IBD-dependent DNA methylation from PBLs appears to have limited ability to detect significant disease associations. More detailed and/or selective approaches may be useful for the elucidation of connections between the DNA methylome and IBD in the future.

Genetic variability in beta-defensins is not associated with susceptibility to Staphylococcus aureus bacteremia.

INTRODUCTION: Human beta-defensins are key components of human innate immunity to a variety of pathogens, including Staphylococcus aureus. The aim of the present study was to investigate a potential association between gene variations in DEFB1 and DEFB103/DEFB4 and the development of S. aureus bacteremia (SAB) employing a case-control design. METHODS: Cases were unique patients with documented SAB, identified with the National S. aureus Bacteremia Register, a comprehensive dataset of all episodes of community associated-SABs (CA-SAB) occurring in children (≤20 yrs) in Denmark from 1990 to 2006. Controls were age-matched healthy individuals with no history of SAB. DNA obtained from cases and controls using the Danish Newborn Screening Biobank were genotyped for functional polymorphisms of DEFB1 by Sanger sequencing and copy number variation of the DEFB103 and DEFB4 genes using Pyrosequencing-based Paralogue Ratio Test (P-PRT). RESULTS: 193 ethnic Danish SAB cases with 382 age-matched controls were used for this study. S. aureus isolates represented a variety of bacterial (i.e., different spa types) types similar to SAB isolates in general. DEFB1 minor allele frequencies of rs11362 (cases vs. controls 0.47/0.44), rs1800972 (0.21/0.24), and rs1799946 (0.32/0.33) were not significantly different in cases compared with controls. Also, DEFB4/DEFB103 gene copy numbers (means 4.83/4.92) were not significantly different in cases compared with controls. CONCLUSIONS: Using a large, unique cohort of pediatric CA-SAB, we found no significant association between DEFB1 genetic variation or DEFB4/DEFB103 gene copy number and susceptibility for SAB.

Mosaicism in segmental Darier disease: an in-depth molecular analysis quantifying proportions of mutated alleles in various tissues.

Darier disease is an autosomal dominant genodermatosis caused by germline mutations in the ATP2A2 gene. Clinical expression is variable, including rare segmental phenotypes thought to be caused by postzygotic mosaicism. Genetic counseling of segmental Darier patients is complex, as risk of transmitting a nonsegmental phenotype to offspring is of unknown magnitude. We present the first in-depth molecular analysis of a mosaic patient with segmental disease, quantifying proportions of mutated and normal alleles in various tissues. Pyrosequence analysis of DNA from semen, affected and normal skin, peripheral leukocytes and hair revealed an uneven distribution of the mutated allele, from 14% in semen to 37% in affected skin. We suggest a model for segmental manifestation expression where a threshold number of mutated cells is needed for manifestation development. We further recommend molecular analysis of the ATP2A2 gene in semen of male patients with segmental Darier disease to improve genetic counseling.

Alpha-defensin DEFA1A3 gene copy number elevation in Danish Crohn's disease patients.

BACKGROUND AND PURPOSE OF STUDY: Extensive copy number variation is observed for the DEFA1A3 gene encoding alpha-defensins 1-3. The objective of this study was to determine the involvement of alpha-defensins in colonic tissue from Crohn's disease (CD) patients and the possible genetic association of DEFA1A3 with CD. METHODS: Two-hundred and forty ethnic Danish CD patients were included in the study. Reverse transcriptase PCR assays determined DEFA1A3 expression in colonic tissue from a subset of patients. Immunohistochemical analysis identified alpha-defensin peptides in colonic tissue. Copy number of DEFA1A3 and individual alleles, DEFA1 and DEFA3, were compared with those for controls, by use of combined real-time quantitative PCR and pyrosequencing, and correlated with disease location. RESULTS: Inflammatory-dependent mRNA expression of DEFA1A3 (P < 0.001), and the presence of alpha-defensin peptides, were observed in colonic tissue samples. Higher DEFA1A3 gene copy number (CD: mean copy number, 7.2 vs. controls 6.7; P < 0.001) and individual DEFA1 alleles (CD mean copy number 5.6 vs. controls 5.1; P < 0.01) were associated with CD, with strong association with colonic location (P < 0.001). CONCLUSIONS: Alpha-defensins are involved in the inflammation of CD, with local mRNA and peptide expression. In combination with the findings that a high DEFA1A3 copy number is significantly linked to CD, these results suggest that a high DEFA1A3 copy number might be important in hindering the normal inflammatory response in CD, particularly colonic CD.