Confirmatory Sanger Sequencing Research Articles

Novel UBE3B mutations: report of eight patients with Kaufman oculocerebrofacial syndrome with additional clinical findings from a highly consanguineous population.

Biallelic mutations in UBE3B cause Kaufman oculocerebrofacial syndrome (KOS; OMIM 244450) with a wide range of clinical manifestations. In this study, we employed genetic analyses including homozygosity mapping, candidate gene sequencing, whole exome sequencing, and confirmatory Sanger sequencing on eight patients from three unrelated consanguineous families. Our analysis yielded three different novel variants in UBE3B : a missense substitution [NM_130466.4: c.2975C>T; (p.Pro992Leu)] in the HECT domain in family 1, a 3-bp deletion within exon 14 [c.1692_1694delCTC; (p.Ser565del)] leading to removal of a serine residue in family 2, and a splice donor site variant in intron eight of UBE3B (c.630 + 1G>T) in family 3. Blepharophimosis, telecanthus, ptosis, intellectual disability and abnormal lipid profile were similar to those found in previously reported KOS patients. Longitudinal follow-up revealed rather marfanoid body habitus of the patients in family 1. This study reports eight patients from Saudi Arabia with novel deleterious variants in UBE3B and adds to the phenotypic spectrum of KOS.

Genome Sequencing of Consanguineous Family Implicates Ubiquitin-Specific Protease 53 (USP53) Variant in Psychosis/Schizophrenia: Wild-Type Expression in Murine Hippocampal CA 1-3 and Granular Dentate with AMPA Synapse Interactions.

Psychosis is a severe mental disorder characterized by abnormal thoughts and perceptions (e.g., hallucinations) occurring quintessentially in schizophrenia and in several other neuropsychiatric disorders. Schizophrenia is widely considered as a neurodevelopmental disorder that onsets during teenage/early adulthood. A multiplex consanguineous Pakistani family was afflicted with severe psychosis and apparent autosomal recessive transmission. The first-cousin parents and five children were healthy, whereas two teenage daughters were severely affected. Structured interviews confirmed the diagnosis of DSM-V schizophrenia. Probands and father underwent next-generation sequencing. All available relatives were subjected to confirmatory Sanger sequencing. Homozygosity mapping and directed a priori filtering identified only one rare variant [MAF < 5(10)-5] at a residue conserved across vertebrates. The variant was a non-catalytic deubiquitinase, USP53 (p.Cys228Arg), predicted in silico as damaging. Genome sequencing did not identify any other potentially pathogenic single nucleotide variant or structural variant. Since the literature on USP53 lacked relevance to mental illness or CNS expression, studies were conducted which revealed USP53 localization in regions of the hippocampus (CA 1-3) and granular dentate. The staining pattern was like that seen with GRIA2/GluA2 and GRIP2 antibodies. All three proteins coimmunoprecipitated. These findings support the glutamate hypothesis of schizophrenia as part of the AMPA-R interactome. If confirmed, USP53 appears to be one of the few Mendelian variants potentially causal to a common-appearing mental disorder that is a rare genetic form of schizophrenia.

Identification of potential susceptibility genes in patients with primary Sjögren’s syndrome-associated pulmonary arterial hypertension through whole exome sequencing

BackgroundPulmonary arterial hypertension (PAH) is a rare complication of primary Sjögren’s syndrome (pSS). Several genes have proven to be associated with pSS and PAH. However, there is no study specifically addressing the genetic susceptibility in pSS combined with PAH.MethodsThirty-four unrelated patients with pSS-PAH were recruited from April 2019 to July 2021 at Peking Union Medical College Hospital. Demographic and clinical data were recorded in detail, and peripheral blood samples were collected for whole-exome sequencing (WES). Gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis were performed to predict the functional effect of mutant genes. Genetic variants identified by WES were confirmed by polymerase chain reaction (PCR)-Sanger sequencing.ResultsWe totally identified 141 pathogenic variant loci of 129 genes in these 34 pSS-PAH patients, using WES analysis. Patients with a family history of rheumatic diseases are more likely to carry FLG mutations or carry gene variations related to the biosynthesis of the amino acids pathway (p < 0.05). According to Sanger sequencing confirmation and pathogenicity validation, we totally identified five candidate pathogenic variants including FLG c.12064A > T, BCR c.3275_3278dupCCGG, GIGYF2 c.3463C > A, ITK c.1741C > T, and SLC26A4 c.919-2A > G.ConclusionOur findings provide preliminary data of exome sequencing to identify susceptibility loci for pSS-PAH and enriched our understanding of the genetic etiology for pSS-PAH. The candidate pathogenic genes may be the potential genetic markers for early warning of this disease.

Genetic variants in the vitamin D pathway and their association with vitamin D metabolite levels: Detailed studies of an inner-city pediatric population suggest a modest but significant effect in early childhood

ObjectivesIn a large cohort of healthy infants and toddlers 6–36 months of age (n = 776), we have been exploring the potential role of genetic variation in predisposition to vitamin D insufficiency. The genes encoding the key cytochrome P450 hydroxylases (CYP2R1, CYP24A1, and CYP27B1) harbour recurrent mutations of uncertain effect. This study was undertaken to look for biochemically relevant associations of these variants with inter-individual differences in vitamin D metabolism in an at-risk pediatric population. MethodsGenotyping for CYP2R1-CT (c.–1127 C>T, rs10741657), CYP24A1-AG (c.–686A>G, rs111622401), and CYP27B1-CA (c.–1261 C>A, rs10877012) mutations were performed using SNaPshot assay, followed by Sanger sequencing confirmation. Vitamin D metabolites and vitamin D binding protein (DBP) were measured by established methods. ResultsIn a multivariate regression model, with corrections for co-variates, subjects with the homozygous CYP2R1-TT variant had significantly higher concentrations of 25(OH)D, free 25(OH)D, and 24,25(OH)2D levels. In subjects with the CYP24A1-AG mutation, concentrations of 25(OH)D were significantly higher. ConclusionsThe CYP2R1-TT and CYP24A1-AG variants have measurable effects on the vitamin D pathway. It seems unlikely that they will be clinically relevant in isolation, but they may be members of the large pool of infrequent mutations contributing to different risks for the vitamin D deficiency phenotype.

CFAP70 is a solid and valuable target for the genetic diagnosis of oligo-astheno-teratozoospermia in infertile men.

Male infertility is a worldwide population health concern, but its aetiology remains largely understood. Although CFAP70 variants have already been reported in two oligo-astheno-teratozoospermia (OAT) individuals by sequencing, animal evidence to support CFAP70 as a credible OAT-pathogenic gene is lacking. Cfap70-KO mice were generated to explore the physiological role of CFAP70. CFAP70 variants were detected in infertile men with OAT by whole exome sequencing and Sanger sequencing confirmation. Cfap70-truncated mice were further generated to explore the pathogenicity of the nonsense variant of CFAP70 identified in the proband. Here, we demonstrate that Cfap70-KO mice are sterile mainly due to OAT and further identify a Chinese infertile man carrying a homozygous nonsense variant (c.2962C>T/p.R988X) of CFAP70. Cfap70-truncated mice lacking 5-8 tetratricopeptide repeats (TPRs) mimic the patient's symptoms. CFAP70 is required for the biogenesis of spermatid flagella partially by regulating the expression of OAT-associated proteins (e.g., QRICH2), assisting the cytoplasmic preassembly of the calmodulin- and radial spoke-associated complex (CSC), and controlling the manchette localization of axoneme-related proteins. Moreover, we suggest that CFAP70-associated male infertility could be overcome by intracytoplasmic sperm injection (ICSI) treatment. Overall, we demonstrate that CFAP70 is necessary to assemble spermatid flagella and that CFAP70 gene could be used as a diagnostic target for male infertility with OAT in the clinic. This study was supported by the National Key Research and Development Project (2019YFA0802101 to S.C), Open Fund of Key Laboratory of Cell Proliferation and Regulation Biology, Ministry of Education (to S.C), Central Government to Guide Local Scientific and Technological Development (ZY21195023 to B.W), and Basic Research Projects of Central Scientific Research Institutes (to B.W).

BG06 Beyond keratin: when simplex is not so simple

Abstract Epidermolysis bullosa simplex (EBS) is a heterogeneous, congenital, mucocutaneous fragility syndrome, characterized ultrastructurally by cleavage within the basal keratinocyte. Seventy-five per cent of cases are attributable to mutations in KRT5 and KRT14, the vast majority of which are dominant negative mutations, resulting in impaired keratin intermediate filament dynamics within the basal layer of the epidermis. The presenting phenotype, including extracutaneous manifestations and overall severity, is often closely linked to the underlying genetic mutation; however, genotype–­phenotype correlation is poorly resolved in a minority of cases, and there is an approximately additional 15% of patients in whom genetic abnormalities are not identified. However, recent advances in next-generation sequencing technologies have expanded the mutational database. We describe two cases of EBS with rare underlying mutations and distinct clinical phenotypes. A 46-year-old man presented with widespread blistering primarily affecting his hands, feet, elbows and knees since childhood. He had unaffected, consanguineous parents and one of his three siblings was affected. Other presenting features included dystrophic nails, focal plantar keratoderma, truncal mottled pigmentation and large areas of hypopigmentation on the back. We considered a diagnosis of ‘EBS with mottled pigmentation’, but the autosomal recessive mode of inheritance and dystrophic nails were atypical. DNA whole-exome sequencing (WES) and bidirectional Sanger sequencing revealed a novel homozygous PLEC mutation [c.94dupG p.(Ala32GlyfsTer3)]. Pathogenic PLEC variants are known to cause autosomal dominant EBS, the Ogna type (MIM131950), autosomal recessive EBS with muscular dystrophy (MIM226670) and autosomal recessive EBS with pyloric atresia (MIM612138). To date, our patient has not manifested any signs of progressive muscle weakness, but the presenting age can vary. The second patient was a 30-year-old woman who presented with blistering on her flexures, feet, hands and friction sites since the age of 4 months. She had unaffected, consanguineous parents and eight unaffected siblings. WES and confirmatory bidirectional Sanger sequencing identified a homozygous nonsense mutation in DST [c.3370C&amp;gt;T p.(Gln1124Ter)]. DST encodes the epithelial isoform of bullous pemphigoid antigen-1, present in the skin, muscle and neuronal tissues. Only 11 patients with DST mutation-induced EBS have been reported to date. The cutaneous phenotype is reported to be blisters, postinflammatory dyspigmentation, scarring, nail dystrophy, keratoderma and hyperhidrosis. If the affected exon is present in neuronal isoforms and neuronal tissue, neurological symptoms are expected. Unlike the patient reported elsewhere who harbours exactly the same mutation, our patient has reported no neurological issues to date. These cases highlight the importance of unravelling genotype–phenotype correlation in rare autosomal recessive forms of EBS, with possible implications for future prognosis and management.

Clinical, radiological, and genetic characterization of SLC13A5 variants in Saudi families: Genotype phenotype correlation and brief review of the literature.

SLC13A5 (solute carrier family 13, member 5) encodes sodium/citrate cotransporter, which mainly localizes in cellular plasma membranes in the frontal cortex, retina, and liver. Pathogenic variants of the gene cause an autosomal recessive syndrome known as "developmental and epileptic encephalopathy 25 with amelogenesis imperfecta." Here, we have investigated six patients from three different consanguineous Saudi families. The affected individuals presented with neonatal seizures, developmental delay, and significant defects in tooth development. Some patients showed other clinical features such as muscle weakness, motor difficulties, intellectual disability, microcephaly, and speech problems in addition to additional abnormalities revealed by electroencephalography (EEGs) and magnetic resonance imaging (MRI). One of the MRI findings was related to cortical thickening in the frontal lobe. To diagnose and study the genetic defects of the patients, whole exome sequencing (WES) coupled with confirmatory Sanger sequencing was utilized. Iterative filtering identified two variants of SLC13A5, one of which is novel, in the families. Families 1 and 2 had the same insertion (a previously reported mutation), leading to a frameshift and premature stop codon. The third family had a novel splice site variant. Confirmatory Sanger sequencing corroborated WES results and indicated full segregation of the variants in the corresponding families. The patients' conditions were poorly controlled by multiple antiepileptics as they needed constant care. Considering that recessive mutations are common in the Arab population, SLC13A5 screening should be prioritized in future patients harboring similar symptoms including defects in molar development.

Cystic neutrophilic granulomatous mastitis: sensitivity and specificity of 16s rRNA and Sanger sequencing for Corynebacterium spp

AimsCystic neutrophilic granulomatous mastitis (CNGM) is a subtype of granulomatous mastitis (GM) associated with Corynebacterium spp infection. We aimed to analyse the prevalence of Corynebacteria in CNGM and non-CNGM cases.MethodsBreast...

ZMYM3 : a new candidate gene in nonobstructive azoospermia?

ZMYM3 : a new candidate gene in nonobstructive azoospermia?

Role of genetic testing in young patients with idiopathic atrioventricular conduction disease.

To investigate the role of genetic testing in patients with idiopathic atrioventricular conduction disease requiring pacemaker (PM) implantation before the age of 50 years. All consecutive PM implantations in Southern Switzerland between 2010 and 2019 were evaluated. Inclusion criteria were: (i) age at the time of PM implantation: < 50 years; (ii) atrioventricular block (AVB) of unknown aetiology. Study population was investigated by ajmaline challenge and echocardiographic assessment over time. Genetic testing was performed using next-generation sequencing panel, containing 174 genes associated to inherited cardiac diseases, and Sanger sequencing confirmation of suspected variants with clinical implication. Of 2510 patients who underwent PM implantation, 15 (0.6%) were young adults (median age: 44 years, male predominance) presenting with advanced AVB of unknown origin. The average incidence of idiopathic AVB computed over the 2010-2019 time window was 0.7 per 100 000 persons per year (95% CI 0.4-1.2). Most of patients (67%) presented with specific genetic findings (pathogenic variant) or variants of uncertain significance (VUS). A pathogenic variant of PKP2 gene was found in one patient (6.7%) with no overt structural cardiac abnormalities. A VUS of TRPM4, MYBPC3, SCN5A, KCNE1, LMNA, GJA5 genes was found in other nine cases (60%). Of these, three unrelated patients (20%) presented the same heterozygous missense variant c.2531G > A p.(Gly844Asp) in TRPM4 gene. Diagnostic re-assessment over time led to a diagnosis of Brugada syndrome and long-QT syndrome in two patients (13%). No cardiac events occurred during a median follow-up of 72 months. Idiopathic AVB in adults younger than 50 years is a very rare condition with an incidence of 0.7 per 100 000 persons/year. Systematic investigations, including genetic testing and ajmaline challenge, can lead to the achievement of a specific diagnosis in up to 20% of patients. Heterozygous missense variant c.2531G > A p.(Gly844Asp) in TRPM4 gene was found in an additional 20% of unrelated patients, suggesting possible association of the variant with the disease.

Biallelic mutations of TTC12 and TTC21B were identified in Chinese patients with multisystem ciliopathy syndromes

BackgroundAbnormalities in cilia ultrastructure and function lead to a range of human phenotypes termed ciliopathies. Many tetratricopeptide repeat domain (TTC) family members have been reported to play critical roles in cilium organization and function.ResultsHere, we describe five unrelated family trios with multisystem ciliopathy syndromes, including situs abnormality, complex congenital heart disease, nephronophthisis or neonatal cholestasis. Through whole-exome sequencing and Sanger sequencing confirmation, we identified compound heterozygous mutations of TTC12 and TTC21B in six affected individuals of Chinese origin. These nonsynonymous mutations affected highly conserved residues and were consistently predicted to be pathogenic. Furthermore, ex vivo cDNA amplification demonstrated that homozygous c.1464 + 2 T > C of TTC12 would cause a whole exon 16 skipping. Both mRNA and protein levels of TTC12 were significantly downregulated in the cells derived from the patient carrying TTC12 mutation c.1464 + 2 T > C by real-time qPCR and immunofluorescence assays when compared with two healthy controls. Transmission electron microscopy analysis further identified ultrastructural defects of the inner dynein arms in this patient. Finally, the effect of TTC12 deficiency on cardiac LR patterning was recapitulated by employing a morpholino-mediated knockdown of ttc12 in zebrafish.ConclusionsTo the best of our knowledge, this is the first study reporting the association between TTC12 variants and ciliopathies in a Chinese population. In addition to nephronophthisis and laterality defects, our findings demonstrated that TTC21B should also be considered a candidate gene for biliary ciliopathy, such as TTC26, which further expands the phenotypic spectrum of TTC21B deficiency in humans.

A Novel Homozygous Founder Variant of RTN4IP1 in Two Consanguineous Saudi Families.

The genetic architecture of mitochondrial disease continues to expand and currently exceeds more than 350 disease-causing genes. Bi-allelic variants in RTN4IP1, also known as Optic Atrophy-10 (OPA10), lead to early-onset recessive optic neuropathy, atrophy, and encephalopathy in the afflicted patients. The gene is known to encode a mitochondrial ubiquinol oxidoreductase that interacts with reticulon 4 and is thought to be a mitochondrial antioxidant NADPH oxidoreductase. Here, we describe two unrelated consanguineous families from the northern region of Saudi Arabia harboring a missense variant (RTN4IP1:NM_032730.5; c.475G<T, p.Val159Phe) in the gene. Clinically affected individuals presented with intellectual disability, encephalopathy, ataxia, optic atrophy, and seizures. Based on whole exome sequencing and confirmatory Sanger sequencing, the variant was fully segregated with the phenotype in the families, absent among large ethnically matching controls as well as numerous in-house exomes, and predicted to be pathogenic by different in silico classifiers. Structural modeling and immunoblot analyses strongly indicated this variant to be pathogenic. Since the families belong to one of the tribal inhabitants of Saudi Arabia, we postulate that the variant is likely to be a founder. We provide the estimated age of the variant and present data confirming the disease-causality of this founder variant.

A novel homozygous ZNF469 variant causing brittle cornea syndrome is associated with corneal ectasias in heterozygous carriers.

To describe a family segregating a novel truncating ZNF469 homozygous mutation causing brittle cornea syndrome type 1 in a male patient and associated with corneal ectasia in his two heterozygous young children. A 49-year-old affected male and his 12- and 8-year-old, apparently healthy, siblings underwent phenotypic and genetic assessment. An Oculus Pentacam Scheimpflug topographer system was employed for keratometries and central corneal thickness measurements. Exome sequencing was performed in DNA from the index case with subsequent Sanger sequencing confirmation of the ZNF469 gene causal variant in his relatives. The index case had a history of bilateral keratoglobus, corneal perforations, bilateral hypoacusia, and skeletal anomalies. His two children exhibited topographic anomalies compatible with keratoconus suspects as well as mild skeletal anomalies. Genetic analysis identified a novel homozygous c.2340delC variant in the ZNF469 gene, which predicts a p.(Arg781Glufs*19) truncated protein. Sanger sequencing identified heterozygosity for the c.2340delC variant in DNA from both siblings. Our results expand the mutational spectrum associated with brittle cornea syndrome and provide the first demonstration of early corneal anomalies in subjects carrying monoallelic ZNF469 variants.

Application of Whole Exome Sequencing in Mutational Analysis of Patients with Ohtahara Syndrome

Ohtahara syndrome is one of the earliest and most severe forms of developmental and epileptic encephalopathy. Over the last decade, the rapid advances in molecular techniques, especially in high-throughput sequencing (HTS), have revealed that a majority of Ohtahara patients have genetic etiology. About 20 genes have been found to be related to this syndrome so far, and Next Generation Sequencing (NGS) technique is now an important genetic test for this syndrome. This study was conducted on 4 patients with Ohtahara syndrome referred to Children’s Hospital 2, Ho Chi Minh City. Whole-exome sequencing (WES) following targeted analysis on 283 epileptic encephalopathy–related genes was performed to identify disease-causing variants of the patients. Following multi-step bioinformatics analysis, trio-based Sanger sequencing confirmation, and variant classification according to standards of The American College of Medical Genetics and Genomics – 2015, we have identified 2 pathogenic mutations in 2 patients: OH3 (KCNQ2, c.868G&gt;A, p.G290S) and OH4 (SCN2A, c.788C&gt;T, p.A263V). The results of this study contribute to verifying the role of genetic factors in Ohtahara syndrome in Vietnamese patients. This study also confirms that NGS in general and WES, in particular, are reliable and useful in detecting genetic causes of Ohtahara syndrome, thereby, assisting in diagnosis and treatment of this syndrome.&#x0D; &#x0D; &#x0D; &#x0D; &#x0D; &#x0D;

FOCAD Indel in a Family With Juvenile Polyposis Syndrome.

Juvenile polyposis syndrome (JPS) is a childhood polyposis syndrome with up to a 50% lifetime risk of gastrointestinal cancer. Germline pathogenic variants in BMPR1A and SMAD4 are responsible for around 40% of cases of JPS, but for the majority of individuals, the underlying genetic cause is unknown. We identified a family for which polyposis spanned four generations, and the proband had a clinical diagnosis of JPS. Next-generation sequencing was conducted, followed by Sanger sequencing confirmation. We identified an internal deletion of the FOCAD gene in all family members tested that altered the reading frame and is predicted to be pathogenic. We conclude that inactivation of the FOCAD gene is likely to cause JPS in this family.

Hyaluronan Synthase 1: A Novel Candidate Gene Associated With Late-Onset Non-syndromic Hereditary Hearing Loss

ObjectivesHyaluronan synthase 1 (HAS1) is a membrane-bound protein that is abundant in the epidermis and dermis, and it is important for skin function. However, its association with hearing loss has not yet been studied. Herein, we sought to evaluate the potential contribution of HAS1: c.1082G>A to genetic hearing loss.MethodsWe used whole-exome sequencing to analyze blood DNA samples of six patients of a family with autosomal dominant familial late-onset progressive hearing loss, which was revealed to be related to a variant of the HAS1 gene. Confirmatory Sanger sequencing was performed with samples from 10 members. A missense variant was detected in HAS1 (c.1082 G>A, p.Cys361Tyr). In silico analyses predicted this variant to result in the functional loss of HAS1. Immunostaining was conducted using wild-type mouse samples to verify HAS1 expression.ResultsHas1 was detected in an otocyst at E10.5. In the pup, Has1 expression was localized in the stria vascularis (SV), hair cells, supporting cells of the organ of Corti, and some spiral ganglion neurons. SV marginal cells markedly expressed Has1 in the adult stage. The hearing threshold in the Has1-depleted condition was investigated by accessing the International Mouse Phenotyping Consortium’s Auditory Brainstem Response (ABR) data. ABR of Has1 knock-out mice showed threshold elevations at 6, 12, and 18 kHz in young male adults.ConclusionHAS1 may have a close relationship with auditory function and genetic hearing loss. Further investigation is needed to reveal the precise role of HAS1 in the auditory system. HAS1 is a candidate gene for future hereditary hearing loss genetic testing.

The Identification of Novel CYP2D6 Variants in US Hmong: Results From Genome Sequencing and Clinical Genotyping.

Objective: Hmong individuals represent a unique East Asian subpopulation in whom limited information concerning pharmacogenetic variation exists. The objectives of this study were to comprehensively characterize the highly polymorphic CYP2D6 gene in Hmong, estimate allele and phenotype frequencies and to compare results between two testing platforms. Methods: DNA from 48 self-identified Hmong participants were sequenced using a targeted next-generation sequencing (NGS) panel. Star allele calls were made using Astrolabe, manual inspection of NGS variant calls and confirmatory Sanger sequencing. Structural variation was determined by long-range (XL)-PCR and digital droplet PCR (ddPCR). The consensus diplotypes were subsequently translated into phenotype utilizing the activity score system. Clinical grade pharmacogenetic testing was obtained for 12 of the 48 samples enabling an assessment of concordance between the consensus calls and those determined by clinical testing platforms. Results: A total of 13 CYP2D6 alleles were identified. The most common alleles were CYP2D6*10 and its structural arrangements (37.5%, 36/96) and the *5 gene deletion (13.5%, 13/96). Three novel suballeles (*10.007, *36.004, and *75.002) were also identified. Phenotype frequencies were as follows: ultrarapid metabolizers (4.2%, 2/48), normal metabolizers (41.7%, 20/48) and intermediate metabolizers (52.1%, 25/48); none of the 48 participants were predicted to be poor metabolizers. Concordance of diplotype and phenotype calls between the consensus and clinical testing were 66.7 and 50%, respectively. Conclusion: Our study to explore CYP2D6 genotypes in the Hmong population suggests that this subpopulation is unique regarding CYP2D6 allelic variants; also, a higher portion of Hmong participants (50%) are predicted to have an intermediate metabolizer phenotype for CYP2D6 compared to other East Asians which range between 27 and 44%. Results from different testing methods varied considerably. These preliminary findings underscore the importance of thoroughly interrogating unique subpopulations to accurately predict a patient’s CYP2D6 metabolizer status.

Molecularly confirmed pontocerebellar hypoplasia in a large family from Slovakia with four severely affected children.

Pontocerebellar hypoplasia type 1 (PCH1) is characterized by a central and peripheral motor dysfunction associated with anterior horn cell degeneration, similar to spinal muscular atrophy (SMA). We analysed three probands (later discovered to be siblings) suspected to have severe SMA, however, not confirmed by genetic test. Clinical-exome analysis (Illumina) was performed to identify causative variants, followed by Sanger sequencing confirmation in probands and other 10 family members. Homozygous pathogenic variant c.92G>C (p.(Gly31Ala)) in the Exosome Component 3 (EXOSC3) gene was found in all 3 probands, thus confirming the diagnosis of a severe form of PCH1B. The parents and six siblings were carriers, while one sibling was homozygous for a reference allele. This variant is frequent in the Czech Roma population, where it is considered a founder mutation. Haplotype analysis in this largest reported PCH1B family showed that our patients inherited from their father (of Roma origin) a haplotype identical to that found in the Czech Roma population, thus indicating these alleles have a common origin. This EXOSC3 variant is rare among the general population but most likely frequent also among Roma people in Slovakia. PCH1B should be considered for a differential diagnosis in infants manifesting SMA-like phenotype, especially if of Roma origin (Tab. 1, Fig. 1, Ref. 22). Text in PDF www.elis.sk Keywords: pontocerebellar hypoplasia, PCH1B, EXOSC3, SMA plus syndromes, pathogenic sequence variant.

Efficacy of combinational therapy using blue light and benzoyl peroxide in reducing Cutibacterium acnes bioburden at the deltopectoral interval: a randomized controlled trial.

The purpose of this study was to compare the efficacy of blue light therapy (BLT) and 5% topical benzoyl peroxide (BPO) gel in combination with standard chlorhexidine (CHX) preparation in eradicating Cutibacterium acnes at the deltopectoral interval measured by positive, quantitative culture findings. Adult male volunteers were randomized to 1 of 3 treatment groups: BPO, BLT, and BPO followed by BLT. Contralateral shoulders served as matched controls. Volunteers randomized to BPO applied the gel for a total of 5 treatments. In the BLT group, a single 23-minute treatment was administered at an estimated irradiance of 40 mW/cm2 (radiant exposure, 55.2 J/cm2). In the BPO-BLT group, volunteers received both treatments as described earlier. After treatment with either BPO, BLT, or both, a single swab culture was taken from the treatment shoulder. Next, control and treatment shoulders were prepared with CHX, and cultures were taken from each shoulder. Cultures were sent for anaerobic quantitative growth analysis with both polymerase chain reaction and Sanger sequencing confirmation of presumptive C acnes colonies. This study enrolled 60 male volunteers, 20 per group, with no loss to follow-up. After treatment but prior to CHX administration, all culture samples in the BPO group and BLT group grew C acnes. Prior to CHX, 16 samples (80%) in the BPO-BLT group grew C acnes. On quantitative analysis, the BPO group and BPO-BLT group had significantly less growth of C acnes compared with the BLT group after treatment but prior to CHX (P < .05 for each). Following CHX administration, the BPO and BPO-BLT groups had significantly fewer positive culture findings (odds ratios of 0.03 and 0.29, respectively) and less quantity of growth compared with their control arms (P < .05). This was not seen in the BLT group. For quantitative between-group analysis, no significant synergistic effects were seen with BPO-BLT compared with BPO alone (P = .688). There was no difference in side effects between groups. The combination of topical BPO and CHX was effective at eliminating C acnes in most cases. BLT alone did not demonstrate effective antimicrobial properties against C acnes at the radiant exposure administered in this study. Combining BPO and BLT did not lead to significant synergistic antimicrobial effects. Both BPO and BLT are safe with few, transient side effects reported. More work is needed to determine whether BLT at higher radiant exposures or serial treatment results in bactericidal effects against C acnes invivo.

A NOVEL MISSENSE MUTATION OUTSIDE DNAJ DOMAIN OF DNAJC21 IS ASSOCIATED WITH SHWACHMAN-DIAMOND SYNDROME

Shwachman-Diamond Syndrome (SDS) and related bone marrow failure disorders are characterized by early onset pancytopenia with a hypocellular bone marrow, short stature, and pancreatic insufficiency, along with an increased risk for myeloid malignancies. Recently, several cases with an SDS-like syndrome have been reported to harbor mutations in the DNAJ domain of DNAJC21 . Here, we report an intriguing case of a 13.5 years-old female born to Turkish consanguineous parents with a novel missense mutation occurring outside the DNAJ domain of the DNAJC21 gene. Whole-exome and Sanger sequencing confirmation revealed a homozygous missense mutation in DNAJC21 gene c.463T>C, p.W155R which was considered as pathogenic in in silico analyses. Initially, this patient's vague and atypical symptoms led to uncertainty of the underlying diagnosis. Upon confirmation of the genetic mutation, a number of functional studies such as diepoxibutane test, proliferation test from peripheral blood mononuclear cells, and cytokinesis-block micronucleus cytome assay performed with the patient cells confirmed the likely diagnosis of an SDS-like syndrome attributable to DNAJC21 dysfunction. Through the analysis of this rare case, we illuminate the pleiotropic features of this unique bone marrow failure syndrome and emphasize the paramount role of genomic testing to discriminate a range of closely related bone marrow failure disorders.