Mild Clinical Phenotype Research Articles

658. Increasing Fetal Hemoglobin Expression With TALEN Induced Small Deletions in Peripheral Blood Stem Cells

Hemoglobinopathies are common and morbid inherited conditions caused by mutations in hemoglobin genes that generate pathologic globin molecules or reduced expression of wild type hemoglobin. A subset of individuals with hemoglobinopathy-associated beta globin mutations have a mild clinical phenotype and are found to carry additional disease modifying mutations including small deletions or point mutations that induce fetal hemoglobin expression. This condition is referred to as hereditary persistence of fetal hemoglobin. Recent progress in the arenas of gene editing and hematopoietic stem cell (HSC) expansion has raised the possibility of treating hemoglobinopathies through direct modification of genes in HSCs to increase the expression of fetal hemoglobin in the erythroid lineage. To date, most attempts to induce fetal hemoglobin have targeted the expression of transcription factors implicated in regulation of fetal hemoglobin production, such as Bcl11a, an approach which could lead to unintended and potentially deleterious effects on cellular phenotype. Here, we are evaluating the application of mRNA transfection of TALENs to directly modify transcription factor binding sites within the hemoglobin locus in HSCs to achieve efficient, stable expression of fetal hemoglobin. We have designed TALENs to induce small deletions in the beta hemoglobin promoter at the binding site of KLF1, postulated to play a role in localizing the active chromatin hub to and preferentially expressing adult beta hemoglobin, as well as other globin locus target regions implicated in the regulation of fetal hemoglobin production. By combining TALEN-mediated gene modification with novel HSC expansion approaches involving the recently described small molecule UM729, we hope to realize both efficient HSC modification and expansion with a sufficient level of fetal hemoglobin production in erythrocytes to achieve a functional cure for a wide range of beta hemoglobinopathies.

Anderson-Fabry: una malattia rara?

Anderson-Fabry: a rare disease? Anderson-Fabry (or Fabry) disease takes the name from the two dermatologists that independently described for the first time the pathology at the end of 1800. It is a multisystemic lysosomal storage disease, inherited in a X-linked manner. Fabry manifestations are slowly progressive, with variable age of onset, severity and clinical course. Usually, in the childhood or in the first period of the adolescence, the patients can manifest angiokeratomas, corneal or lenticular opacity, microalbuminuria or proteinuria and symptoms reflecting the involvement of peripheral nervous system, like episodic crises of acute pain, acroparesthesias, anhidrosis or hypohidrosis. With the age, there is a progression of the disease with a gradual deterioration of kidney function, that leads to End Stage Renal Disease (ESRD), and the onset of severe cardiovascular and cerebrovascular complications, that cause a reduction in quality of life in affected patients. Such complications, or a combination of these, can lead to the premature death of the patient within the fourth or fifth decade of life. In addition to the classic form, atypical variants of the disease were described: these variants manifest a mild and late-onset clinical phenotype, that is difficult to diagnose because of the involvement of a single organ. The 80% of Fabry patients are affected by these variants with a non-specific symptomatology that makes the diagnosis, which can be made in the adulthood after several years since the manifestation of the first symptoms, when the internal organs are irreversibly compromised, difficult. It was estimated a considerable delay in the diagnosis in about 40% of male patients and 70% of female patients. In particular, there is a time interval of 13 years for men and 17 years for women between the onset of the first symptoms and the correct diagnosis. Therefore, Fabry disease should be considered among the diagnostic hypothesis in all the subjects that present a systemic clinical course referable to the disease. The availability of recombinant enzyme replacement therapy has an important impact on clinical management of affected patients, improving the prognosis and the quality of life. The data in literature show the importance of a specific therapeutic intervention made as early as possible, before an irreversible organ involvement.

Overdominance Effects between Malaria and Visceral Leishmaniasis in the 5q31 Region

A group of SNPs in 5q31, genotyped from Hausa tribe of Koka village in eastern Sudan; an area endemic with malaria and visceral leishmaniasis, were analyzed by The Leishmaniasis research group and found to have significant excess of heterozygosis level and departure from HWE and assumed to be due to natural selection resulting from notorious deadly outbreaks since various ethnic groups from western Sudan settled the area although sequence information in the 5q31 region did not detect functional SNPs that could be associated with such drastic phenotypes. Malaria was thought to inflict inferior selective pressure due to the mild clinical phenotype observed. Taking advantage of follow up phenotype data sets available from the Malaria Gen study we re-analyzed these genotypes using different bioinformatics software to determine their effect on the regulation, function and expression of interleukins, miRNA binding and splicing mechanism. The SNPs were found to potentially affect the binding of many transcription factors that regulate the expression of IL-4 and IL-13 and stability of IL-5 mRNA. Association of haplotypes susceptibility revealed that the haplotype of low cytokine TH2 profile is associated with higher risk of malaria infection (P-value = 0.02). Through modulating TH2 cytokine response; the excess heterozygosity in 5q31 is explained with the phenomenon of overdominance between malaria and visceral leishmaniasis, acted by natural selection and driving the locus towards optimum response.

The molecular basis of low activity levels of coagulation factor VII: a Brazilian cohort.

Inherited factor VII (FVII) deficiency is the most common among the rare bleeding disorders. It is transmitted as an autosomal recessive inheritance, due to mutations in the FVII gene (F7). Molecular studies of FVII deficiency are rare in non-Caucasian populations. The aim of the study was to evaluate the molecular basis behind low levels of FVII activity (FVII:C) levelsin a cohort of Brazilian patients. A total of 34 patients with low FVII levels were clinically evaluated and submitted to laboratory tests, among these, prothrombin time and FVII:C, with different thromboplastins. All exons and intron/exon boundaries of F7 were amplified and sequenced. A total of 14 genetic alterations were identified, of which six were described previously, c.1091G>A, c.1151C>T, c.-323_-313insCCTATATCCT, c.285G>A, c.525C>T, c.1238G>A and eight (54.0%) and eight were new, c.128G>A, c.252C>T, c.348G>A, c.417G>A, c.426G>A, c.745_747delGTG, c.843G>A and c.805+52C>T. In addition to the mutation c.1091G>A, known as FVII Padua, the mutation c.1151C>T also presented discrepant FVII:C levels when tested with human and rabbit brain thromboplastin. There was no association between phenotype and genotype. Most of the identified genetic alterations found were polymorphisms. Low levels of FVII:C in this population were mostly related to polymorphisms in F7 and associated with a mild clinical phenotype. Mutation c.1151C>T was associated with discrepant levels of FVII:C using different thromboplastins, such as reported with FVII Padua.

Biochemical and computational analyses of two phenotypically related GALT mutations (S222N and S135L) that lead to atypical galactosemia.

Galactosemia is a metabolic disorder caused by mutations in the GALT gene [1,2]. We encountered a patient heterozygous for a known pathogenic H132Q mutation and a novel S222N variant of unknown significance [3]. Reminiscent of patients with the S135L mutation, our patient had loss of GALT enzyme activity in erythrocytes but a very mild clinical phenotype [3–8]. We performed splicing experiments and computational structural analyses to investigate the role of the novel S222N variant. Alamut software data predicted loss of splicing enhancers for the S222N and S135L mutations [9,10]. A cDNA library was generated from our patient׳s RNA to investigate for splicing errors, but no change in transcript length was seen [3]. In silico structural analysis was performed to investigate enzyme stability and attempt to understand the mechanism of the atypical galactosemia phenotype. Stability results are publicly available in the GALT Protein Database 2.0 [11–14]. Animations were created to give the reader a dynamic view of the enzyme structure and mutation locations. Protein database files and python scripts are included for further investigation.

The Platelet-Derived Microparticles Related to the Clinical Phenotype Heterogeneity of Hemophlia a

Keywords: Hemophlia A, Phenotype; Heterogeneity; Flowcytometry ; MircoparticleBackground: The clinical phenotype for hemophlia A are typically recognized sub-clinical, mild, moderate and severe according to the residue FVIII level of the plasma. For recent years, there are evidences indicate that the residue FVIII level is not the only factor relate to the clinical phenotype of patients with severe hemophlia A. Platelets are an important role in hemostasis and platelet-derived microparticles (PMPs) are known to mediate a prothrombotic state in patients, we hypothesized that PMPs contributes to the clinical phenotype heterogeneity of hemophilia A patients.Methods: Thirty-one patients with severe hemophlia A (FVIII:C<1%, age 18-33 yrs, median 23.5 yrs) treated on demand were enrolled in this study, including 10 patients with mild type(FVIII:C<1%, but annualized bleed rates<6) and 21 patients with severe type (FVIII:C<1% and annualized bleed rates≥24). Seventeen healthy male donors were selected as normal controls. We also compared the baseline PMPs counts from eleven patients on demand treatment to that when they had three months prophylactic treatment. Venous blood was anticoagulated with sodium citrate (1:9). Plasma was isolated (1550g for 20 min at RT) and then cell free supernatant was ultracentrifuged (18,000g for 20 min at 20°C). The supernatant plasma samples were measured for baseline PMPs counts, with phycoerythrin (PE)-conjugated monoclonal antibodies CD41, CD42b, CD61, CD62P, and CD63 specific for PMPs derived from platelets respectively. Analyzed by flow cytometry, Forward scatter was set in scale using fluorescent microspheres of 1.0μm and standard fluorescent microbeads (0-1.0μm) in diameter were used to set the microparticle gate. Statistics were performed using independent-samples T testResults: There was a trend toward higher the mean level of PMPs counts in mild type patients compared to severe type (mean ± SD: 2.06±1.33 vs 0.95±0.64, p = 0.02), the PMPs counts of normal individuals was significantly higher than severe type(mean ± SD: 1.06±1.20 v s0.95±0.64, p = 0.02), significantly lower than mild type (mean ± SD: 1.06±1.20 vs 2.06±1.33, p = 0.02). The PMPs counts decreased after 3 months low dose (10 iu/kg, 2-3/w) prophylacticlly treatment (mean ± SD: 6.50±0.51 vs 0.45±0.50, p = 0.00).Conclusion/Discussion: Platelet-derived Microparticles (PMPs) are generated in response to platelet activation, our observations demonstrate that the PMPs counts differs in patients of severe hemophlia A, the mean lever of PMPs in mild clinical phenotype patients was higher than the severe type, prophylacticlly treated patients has a lower PMPs lever, PMPs is likely to play a role in clincal phenotype heterogeneity of sever hemophilia A. DisclosuresNo relevant conflicts of interest to declare.

Mild Clinical Features and Histopathologically Atypical Cores in Two Korean Families with Central Core Disease HarboringRYR1Mutations at the C-Terminal Region

BackgroundCentral core disease (CCD) is a congenital myopathy characterized by distinctive cores in muscle fibers. Mutations in the gene encoding ryanodine receptor 1 (RYR1) have been identified in most CCD patients.Case ReportTwo unrelated patients presented with slowly progressive or nonprogressive proximal muscle weakness since childhood. Their family history revealed some members with the same clinical problem. Histological analysis of muscle biopsy samples revealed numerous peripheral cores in the muscle fibers. RYR1 sequence analysis disclosed a novel mutation in exon 101 (c.14590T>C) and confirmed a previously reported mutation in exon 102 (c.14678G>A).ConclusionsWe report herein two families with CCD in whom missense mutations at the C-terminal of RYR1 were identified. Although it has been accepted that such mutations are usually associated with a severe clinical phenotype and clearly demarcated central cores, our patients exhibited a mild clinical phenotype without facial muscle involvement and skeletal deformities, and atypical cores in their muscle biopsy specimens.

Ataxia telangiectasia: more variation at clinical and cellular levels.

Ataxia telangiectasia (A-T) is a rare recessively inherited disorder resulting in a progressive neurological decline. It is caused by biallelic mutation of the ATM gene that encodes a 370 kDa serine/threonine protein kinase responsible for phosphorylating many target proteins. ATM is activated by auto(trans)phosphorylation in response to DNA double strand breaks and leads to the activation of cell cycle checkpoints and either DNA repair or apoptosis as part of the cellular response to DNA damage. The allelic heterogeneity in A-T is striking. While the majority of mutations are truncating, leading to instability and loss of the ATM protein from the allele, a significant proportion of patients carry one of a small number of mutations that are either missense or leaky splice site mutations resulting in retention of some ATM with activity. The allelic heterogeneity in ATM, therefore, results in an equally striking clinical heterogeneity. There is also locus heterogeneity because mutation of the MRE11 gene can cause an obvious A-T like disorder both clinically and also at the cellular level and mutation of the RNF168 gene results in a much milder clinical phenotype, neurologically, with the major clinical feature being an immunological defect.

G.P.42: Mild clinical phenotype in two siblings carrying myotubular myopathy

Among congenital myopathies, X-linked recessive myotubular myopathy is one of the most severe forms. The majority of patients present with severe hypotonia at birth and respiratory insufficiency and most of them die in the first year of life. However, there is phenotypic variability. Here, we report two patients carrying a missense mutation in MTM1 gene and presenting with a mild phenotype. The first boy was born on time with respiratory insufficiency and generalised hypotonia. He required ventilator support for 3 weeks, and then he went home without specific care. He walked at 11 month. He is now 24 and can run, practices canoe and works as a salesperson. At the exam, he presents mild proximal weakness and restrictive syndrome with no ventilator support (VC = 57%). His brother was born on time with respiratory insufficiency and hypotonia. He stayed in intensive care unit for 6 weeks, then went home without support. He walked at 18 months. Respiratory involvement was more severe than his brother with a severe restrictive syndrome (VC = 33%) but the young man refused non-invasive night-time ventilation proposed at the age of 16. Being now 20, he can walk without any difficulty but cannot run. Clinical exam reveals a mild proximal weakness. Both patients underwent muscle biopsy showing the typical centronuclear myopathy pattern. Molecular analysis of MTM1gene found a hemizygous mutation c.575A>G in exon 8 leading to a missense p.Tyr192Cys. A decreased level of the MTM1 protein was observed by western blot analysis on lymphoblasts. To date, about 300 different mutations in around 540 families have been identified in MTM1 gene, including point mutations, insertions and small and large deletions and duplication. Around 70% of patients presenting with a mild or moderate phenotype carries a missense mutation, which is also the most frequent type of mutation. Another patient has been described with the same mutation (c.575A>G) and presented also with a mild phenotype.

Fusobacterium detected in colonic biopsy and clinicopathological features of ulcerative colitis in Japan.

Fusobacterium species are part of the gut microbiome in humans, but some species have been recognized as opportunistic pathogens implicated in inflammatory diseases including inflammatory bowel diseases. Here, we performed prevalence screening of Fusobacterium in ulcerative colitis (UC) in Japanese patients. We examined Fusobacterium nucleatum (F. nucleatum) and whole Fusobacterium species (Pan-fusobacterium) by quantitative real-time PCR in 163 inflamed mucosae from 152 UC patients. Data were correlated with clinical subtypes of UC. In an initial prevalence screen, F. nucleatum and Pan-fusobacterium were detected in 6.3 % (4/64) and 53.1 % (34/64). For all 163 mucosae, the prevalence of Pan-fusobacterium was 54.6 % (89/163). Pan-fusobacterium status was concordant in inflamed and normal adjacent samples, and the matched cases during 1-year follow-up colonoscopy. The higher amount of Pan-fusobacterium was observed in chronic continuous type compared to one attack and relapse/remitting type (p = 0.039). The higher amount of Pan-fusobacterium was also associated with rather mild clinical course of disease, such as non-steroid dependency (p = 0.015), non-refractory phenotype (p = 0.013), and non-severe phenotype (p = 0.04). Based on the distribution of Pan-fusobacterium measurable cases, we identified 10 cases as having a high amount of Pan-fusobacterium (FB-high). The clinicopathological features of FB-high UC cases were also highlighted by chronic continuous type and mild phenotypes of disease. Whole Fusobacterium species, but not F. nucleatum, are common in UC patients and have a role in persistence of colonic inflammation in UC. However, Fusobacterium infection is associated with rather mild clinical phenotypes of UC.

Clinical and molecular characteristics of Chinese patients with X-linked lymphoproliferative syndrome type 1.

X-linked lymphoproliferative syndrome type 1 (XLP1) is a rare inherited, life-threatening immunodeficiency disorder caused by mutations in SH2D1A gene. It affect approximately two to three males per million. Fewer than 10 cases with definite gene mutations have been reported in Chinese mainland and no rapid diagnosis method has been established. We determined the clinical and molecular characteristics of five patients with XLP1. The SH2D1A gene were amplified by PCR and sequenced, the SAP expression was analyzed by flow cytometry. Two patients had novel SH2D1A mutations and three had mutations that have been previously reported. Three patients presented with fulminant infectious mononucleosis or hemophagocytic lymphohistiocytosis and one presented with lymphoma. Null or decreased SAP expression on PBMCs was noted. The remaining patient presented with unique, recurrent, nonfulminant infectious mononucleosis and bimodal intracellular SAP protein expression. The overall molecular characteristics and clinical phenotypes of Chinese patients with XLP1 matched previous reports. The unique bimodal intracellular SAP protein expression indicated the presence of some residual SAP-positive T cells that are able to respond to persistent Epstein-Barr virus infection and could explain the relatively mild clinical phenotype of this patient.

A recurrent deletion syndrome at chromosome bands 2p11.2-2p12 flanked by segmental duplications at the breakpoints and including REEP1.

We identified an identical and recurrent 9.4-Mbp deletion at chromosome bands 2p11.2-2p12, which occurred de novo in two unrelated patients. It is flanked at the distal and proximal breakpoints by two homologous segmental duplications consisting of low copy repeat (LCR) blocks in direct orientation, which have >99% sequence identity. Despite the fact that the deletion was almost 10 Mbp in size, the patients showed a relatively mild clinical phenotype, that is, mild-to-moderate intellectual disability, a happy disposition, speech delay and delayed motor development. Their phenotype matches with that of previously described patients. The 2p11.2-2p12 deletion includes the REEP1 gene that is associated with spastic paraplegia and phenotypic features related to this are apparent in most 2p11.2-2p12 deletion patients, but not in all. Other hemizygous genes that may contribute to the clinical phenotype include LRRTM1 and CTNNA2. We propose a recurrent but rare 2p11.2-2p12 deletion syndrome based on (1) the identical, non-random localisation of the de novo deletion breakpoints in two unrelated patients and a patient from literature, (2) the patients' phenotypic similarity and their phenotypic overlap with other 2p deletions and (3) the presence of highly identical LCR blocks flanking both breakpoints, consistent with a non-allelic homologous recombination (NAHR)-mediated rearrangement.

Effect of RANTES gene promoter genotypes in patients with ulcerative colitis.

A complex interaction of genetic and environmental factors is closely associated with the development of inflammatory bowel disease. Previous studies reported that the expression of the regulated upon activation, normal T-cell expressed and secreted (RANTES) gene is enhanced in the colonic mucosa of ulcerative colitis (UC). Quantitative differences in RANTES gene expression among numerous promoter genotypes have also been reported. The aim of the present study was to clarify the effect of RANTES promoter polymorphism on the risk of UC, including its clinical phenotypes. A total of 150 UC patients and 372 healthy control (HC) subjects participated in the study. The UC patients were classified by disease behavior, severity and extent of disease. Restriction fragment length polymorphism analysis was performed for polymorphisms at -28 C/G in the RANTES gene promoter region. Although no significant difference of the RANTES promoter genotype distribution was observed between the HC and UC groups, the G/G genotype was significantly higher among female (OR=3.95, 95% CI=1.22-12.82, P=0.03), non-steroid dependent (OR=3.37, 95% CI=1.16-9.85, P=0.03) and non-refractory (OR=3.76, 95% CI=1.29-10.98, P=0.02) UC patients. The G carrier was also found to be associated with an increased risk of rectal colitis (OR=2.21, 95% CI=1.12-4.39, P=0.03). The data indicate that the polymorphism of the RANTES promoter is not directly associated with the susceptibility to UC, but the -28 G allele is associated with female UC patients and mild clinical phenotypes of UC, including non-steroid dependency, non-refractory and rectal colitis.

Novel physiological RECQL4 alternative transcript disclosed by molecular characterisation of Rothmund–Thomson Syndrome sibs with mild phenotype

Rothmund-Thomson syndrome is a rare genodermatosis caused by biallelic mutations of the RECQL4 gene and is characterised by poikiloderma, sparse hair, eyelashes and/or eyebrows, small stature, skeletal and dental abnormalities and cancer predisposition. Mutations predicted to result in the loss of RECQL4 protein have been associated with osteosarcoma risk, but mutation(s)-phenotype correlations are better addressed by combined DNA and RNA analyses. We describe two siblings with a mild phenotype, mainly restricted to the skin, who carry the unreported paternal c.2272C>T alteration in exon 14 and the previously reported maternal exon 15 c.2492_2493delAT, both predicted to result in premature termination codons (p.(Arg758*), p.(His831Argfs*52)). However real-time and transcript analysis showed, in the carrier father and affected daughter, increased levels of a novel RECQL4 physiological alternative transcript with partial in-frame skipping of exon 14, generated by increased usage of a weak cryptic splice site. This alternative transcript is expressed in all controls and tested tissues, its upregulation is specific to the paternal c.2272C>T mutation and depends on the abrogation of the binding motifs for SF2 and SRp55 serine/arginine-rich proteins with bypass of the mutation site located in the skipped exon 14 portion. Moreover, in the proband the increased levels of the alternative transcript, likely encoding a protein isoform with residual activity, may compensate for the dearth of the canonical transcript with the c.2492_2493delAT, accounting for the mild clinical phenotype of the siblings. Our results emphasise the value of RNA analysis to better predict the effects of RECQL4 mutations on the clinical phenotype.

Novel LAMB3 mutations cause non‐syndromic amelogenesis imperfecta with variable expressivity

Amelogenesis imperfecta (AI) is a collective term referring to inherited malformation of tooth enamel without other non-oral symptoms. Junctional epidermolysis bullosa (JEB) is a group of recessive genetic disorders featuring skin fragility, easy blistering and AI. Carriers having only one defective allele usually have no disease phenotype; however, rarely, heterozygous conditions can cause autosomal-dominant AI (ADAI) with no or very mild skin fragility. Recently, LAMB3 mutations causing non-syndromic AI have been identified in three families with an autosomal-dominant inheritance pattern 1, 2. We recruited two families with generalized hypoplastic AI without any other medical conditions. The proband of family 1 was a 2-year-old girl presenting with generalized, irregular hypoplastic enamel in all her primary teeth. Newly erupted primary second molars showed small islands of enamel on the cusp tips and cervical areas. She inherited enamel hypoplasia from her mother. Other family members (a grandmother and uncle) were also affected similarly (Fig. 1a–d). The proband of family 2 was an 8-year-old boy who also presented with enamel hypoplasia in all his primary and permanent teeth. Thermal sensitivity was not severe and hypoplastic grooves and pits accentuated the mamelon structures especially in the mandibular incisors. Interestingly, it was reported by his father that no one else in this family was affected by enamel defects. However, close examination of the father of the proband revealed hypoplastic grooves in several teeth (Fig. 1e–i). Whole exome sequencings identified novel frameshift LAMB3 mutations (NM_000228.2: c.3357_3358insC in family 1 and c.3463_3475delGAGCAGATCCGTG in family 2). The mutation in family 1 (c.3357_3358insC) was located only 25 bp away from the splicing donor site of exon 22. A shift in the reading frame would not generate an early termination codon within this exon and would not affect normal mRNA splicing. Therefore, the mutant transcript would escape non-sense-mediated mRNA decay (NMD) and generate a truncated protein with 39 novel amino acids instead of a C-terminal with 53 amino acids in the wild-type LAMB3 (p.Met1120fs*40). The mutation in family 2 (c.3463_3475delGAGCAGATCCGTG) would also escape NMD because of its location in the last exon producing a truncated protein with 50 novel amino acids replacing the C-terminal 18 amino acids of the wild-type LAMB3 (p.Glu1155fs*51) (Fig. 1J). The extremely mild clinical phenotype in the father and grandmother could be caused by the fact that the mutation replaced only 18 amino acids in the C-terminus. JEB is caused by mutations in LAMA3, LAMB3, LAMC2, and COL17A1. Among them, LAMB3 mutations are responsible for about 80% of all JEB cases. Laminin-332 is a basement membrane protein and a heterotrimer composed of three subunits encoded by the LAMA3, LAMB3, and LAMC2 3. Even though mutations in both alleles cause JEB with AI, a single defective allele in a carrier of JEB could result in AI with little or no skin phenotype. Two cases in COL17A1 and one case in LAMA3 heterozygous carrier have been reported to have ADAI with no or a mild skin phenotype 4, 5. Recently, a heterozygous LAMB3 mutation (c.3392_3393insG) has been reported to cause ADAI in a family 1. Subsequently, two more mutations (c.3446_3453delGACTGGAG and c.3431C>A) have been identified to cause ADAI in two families 2. All three mutations are non-sense or frameshift mutations, but the truncated proteins are expected to be expressed escaping NMD because of their location on the last exon. Two novel LAMB3 mutations identified in this study are also expected to escape NMD and be expressed as truncated proteins. Interestingly, highly variable expressivity was confirmed in family 2, while all the affected members of family 1 exhibited consistently a similar hypoplastic enamel phenotype. The mutation in family 2 replaces only 18 amino acids at the C-terminus and produces 1154 amino acids of wild-type LAMB3. This small replacement would explain the phenotypic variation in family 2 (Fig. 1j). The authors are grateful to all family members who participated in this study. This work was supported by grants from the Bio & Medical Technology Development Program (2013037491), the Science Research Center grant to Bone Metabolism Research Center (2008-0062614) by the Korea Research Foundation Grant funded by the Korean government (MEST). K.-E. Leea J. Koa C.G. Tran Lea T.J. Shina H.-K. Hyuna S.-H. Leea J.-W. Kima,b aDepartment of Pediatric Dentistry & Dental Research Institute, School of Dentistry Seoul National University, Seoul, South Korea bDepartment of Molecular Genetics & Dental Research Institute School of Dentistry, Seoul National University, Seoul, South Korea

Adult phenotype and further phenotypic variability in SRD5A3-CDG

BackgroundSRD5A3 is responsible for SRD5A3-CDG, a type of congenital disorder of glycosylation, and mutations have been reported in 15 children. All the mutations are recessive and truncating.Case presentationWe present 2 brothers at the age of 38 and 40 years with an initial diagnosis of cerebellar ataxia. We found the candidate disease loci via linkage analysis using data from single nucleotide polymorphism genome scans and homozygous truncating mutation SRD5A3 p.W19X, which was previously reported in 3 unrelated children, by exome sequencing.Clinical investigations included physical and ocular examinations and blood tests. Severe ocular involvement with retinal bone spicule pigmentation and optic atrophy are the most prominent disabling clinical features of the disease. The serum transferrin isoelectric focusing (TIEF) pattern is abnormal in the patient investigated.ConclusionOur patients are older, with later onset and milder clinical phenotypes than all patients with SRD5A3-CDG reported so far. They also have atypical ocular findings and variable phenotypes. Our findings widen the spectrum of phenotypes resulting from SRD5A3 mutations and the clinical variability of SRD5A3-CDG, and suggest screening for SRD5A3 mutations in new patients with at least a few of the clinical symptoms of SRD5A3-CDG.

A novel KCNQ1 splicing mutation in patients with forme fruste LQT1 aggravated by hypokalemia

BackgroundSeveral KCNQ1 splicing mutations have been identified in patients with type-1 long QT syndrome (LQT1). It was suggested that the clinical severity may differ according to the aberrant splicing products. There may be precipitating factors that cause cardiac events in those with a mild clinical phenotype (forme fruste LQT1). Methods and resultsWe analyzed the KCNQ1, KCNH2, SCN5A, KCNE1, and KCNE2 genes in 31 consecutive LQTS patients. A novel KCNQ1 1251+1G>A (IVS9+1G>A) mutation was identified in three probands and their two relatives. The QT interval in all of the five individuals with mutation was not much prolonged in the absence of precipitating factors (mean QTc was 461±30ms.). Two of the five individuals with mutation were symptomatic. One patient (a 38-year-old female) had experienced recurrent episodes of syncope due to ventricular tachyarrhythmias (VTAs) accompanied by QT prolongation (QTc: 750ms) when the serum potassium concentration ([K+]) was 2.7mEq/L. After correction of [K+], the QTc interval was shortened to 515ms, and the occurrence of VTAs ceased. Another patient (a 22-year-old female) was resuscitated from cardio-pulmonary arrest due to VTAs. Just after resuscitation, the QTc interval was 629ms, and [K+] was 2.9mEq/L. After correction of [K+], the QTc interval was dramatically shortened to 440ms. In order to identify abnormal splicing products of the responsible mutation, we analyzed the reverse transcription-polymerase chain reaction products from peripheral bloods of the mutation carrier, and identified exon 9-skipping (Δ9) and cryptic sequential exons 8 and 9-skipping (Δ8–9) products, as well as a no exon-skipping product. ConclusionsWe identified a novel KCNQ splicing mutation 1251+1G>A in forme fruste LQT1, which induces cryptic splicing. Two of the five individuals with mutation experienced VTAs in the setting of hypokalemia, emphasizing the need to increase awareness of the significance of hypokalemia in this subgroup of LQT1 patients.

COX10Mutations Resulting in Complex Multisystem Mitochondrial Disease That Remains Stable Into Adulthood

Isolated cytochrome-c oxidase (COX) deficiency is one of the most frequent respiratory chain defects seen in human mitochondrial disease. Typically, patients present with severe neonatal multisystem disease and have an early fatal outcome. We describe an adult patient with isolated COX deficiency associated with a relatively mild clinical phenotype comprising myopathy; demyelinating neuropathy; premature ovarian failure; short stature; hearing loss; pigmentary maculopathy; and renal tubular dysfunction. Whole-exome sequencing detected 1 known pathogenic and 1 novel COX10 mutation: c.1007A>T; p.Asp336Val, previously associated with fatal infantile COX deficiency, and c.1015C>T; p.Arg339Trp. Muscle COX holoenzyme and subassemblies were undetectable on immunoblots of blue-native gels, whereas denaturing gels and immunocytochemistry showed reduced core subunit MTCO1. Heme absorption spectra revealed low heme aa3 compatible with heme A:farnesyltransferase deficiency due to COX10 dysfunction. Both mutations demonstrated respiratory deficiency in yeast, confirming pathogenicity. A COX10 protein model was used to predict the structural consequences of the novel Arg339Trp and all previously reported substitutions. These findings establish that COX10 mutations cause adult mitochondrial disease. Nuclear modifiers, epigenetic phenomenon, and/or environmental factors may influence the disease phenotype caused by reduced COX activity and contribute to the variable clinical severity related to COX10 dysfunction.

P295 – 1801 A single aminoacid deletion in the dystrophin protein associated with a mild clinical phenotype

P295 – 1801 A single aminoacid deletion in the dystrophin protein associated with a mild clinical phenotype

Circulation Editors’ Picks

<i>Circulation</i> Editors’ Picks