SLC25A13 Mutations Research Articles

Expanding the phenotype of de novo SLC25A4-linked mitochondrial disease to include mild myopathy.

ObjectiveTo determine the disease relevance of a novel de novo dominant variant in the SLC25A4 gene, encoding the muscle mitochondrial adenosine diphosphate (ADP)/adenosine triphosphate (ATP) carrier, identified in a child presenting with a previously unreported phenotype of mild childhood-onset myopathy.MethodsImmunohistochemical and western blot analysis of the patient's muscle tissue were used to assay for the evidence of mitochondrial myopathy and for complex I–V protein levels. To determine the effect of a putative pathogenic p.Lys33Gln variant on ADP/ATP transport, the mutant protein was expressed in Lactococcus lactis and its transport activity was assessed with fused membrane vesicles.ResultsOur data demonstrate that the heterozygous c.97A>T (p.Lys33Gln) SLC25A4 variant is associated with classic muscle biopsy findings of mitochondrial myopathy (cytochrome c oxidase [COX]-deficient and ragged blue fibers), significantly impaired ADP/ATP transport in Lactococcus lactis and decreased complex I, III, and IV protein levels in patient's skeletal muscle. Nonetheless, the expression levels of the total ADP/ATP carrier (AAC) content in the muscle biopsy was largely unaffected.ConclusionsThis report further expands the clinical phenotype of de novo dominant SLC25A4 mutations to a childhood-onset, mild skeletal myopathy, without evidence of previously reported clinical features associated with SLC25A4-associated disease, such as cardiomyopathy, encephalopathy or ophthalmoplegia. The most likely reason for the milder disease phenotype is that the overall AAC expression levels were not affected, meaning that expression of the wild-type allele and other isoforms may in part have compensated for the impaired mutant variant.

Phenotypic spectrum of SLC25A4 mutations.

There is no comprehensive overview concerning the phenotypic variability in patients carrying SLC25A4 mutations available. Therefore, the aim of the present review was to summarise and discuss recent findings concerning the clinical presentation and phenotypic heterogeneity of SLC25A4 mutations. The study was conducted by systematically reviewing the literature using the search terms 'mitochondrial', "myopathy', 'nuclear DNA', 'mitochondrial DNA', in combination with 'SLC25A4' or 'AAC1'. The results indicated that the phenotypic heterogeneity in patients carrying a SLC25A4 mutation is broader than so far anticipated. Patients carrying a SLC25A4 mutation not only manifest as encephalo-myo-cardiomyopathy but also with scoliosis, cataract, depression, headache, hydrocephalus or arterial hypertension. SLC25A4 mutations may result in mtDNA depletion or multiple mitochondrial (mt)DNA deletions. SLC25A4-associated mtDNA depletion presents with the more severe phenotype and the worse outcome than patients with multiple mtDNA deletions. Depletion syndrome due to SLC25A4 mutations is associated with congenital respiratory insufficiency requiring mechanical ventilation with poor prognosis in the majority of the cases. Mutations in the SLC25A4 gene manifest phenotypically with multiorgan abnormalities in addition to encephalo-myo-cardiomyopathy. SLC25A4 mutations, causing mtDNA depletion, present with a more severe phenotype, including respiratory insufficiency and more widespread cerebral disease than mutations causing multiple mtDNA deletions.

Rapid screening of c. 166A>G in SLC4A1 gene by high-resolution melting analysis

Objective To explore the feasibility of a high-resolution melting (HRM) method for rapid screening of SLC4A1 mutation. Methods Two hereditary spherocytosis (HS) with a c. 166A>G heterozygous mutation of SLC4A1 confirmed by DNA sequencing and thirty healthy controls were selected for the study. The HRM primer was designed by Primer Premier 6.0 software. All of these samples were detected by LightCycler®480 and analyzed by HRM. Results The HRM analysis was able to detect the c. 166A>G heterozygous mutation of SLC4A1 effectively, and the specificity and sensitivity were both 100%. Conclusions The HRM analysis was appropriate for the detection of c. 166A>G in SLC4A1. It was an efficient, accurate and cost-effective molecular diagnosis method.(Chin J Lab Med, 2018, 41: 370-373) Key words: Spherocytosis, hereditary; Anion exchange protein 1, erythrocyte; High resolution melting

Distal renal tubular acidosis. Clinical manifestations in patients with different underlying gene mutations.

To evaluate whether there are differences in the phenotype of primary distal renal tubular acidosis (dRTA) patients according to the causal defective gene. Twenty-seven non-oriental patients with genetically confirmed dRTA were grouped according to the identified underlying mutations in either ATP6V1B1 (n= 10), ATP6V0A4 (n= 12), or SLC4A1 (n= 5) gene. Demographic features, growth impairment, biochemical variables and presence of deafness, nephrocalcinosis, and urolithiasis at diagnosis were compared among the three groups. Patients with SLC4A1 mutations presented later than those with ATP6V1B1 or ATP6V0A4 defects (120 vs. 7 and 3months, respectively). Hearing loss at diagnosis was present in the majority of patients with ATP6V1B1 mutations, in two patients with ATP6V0A4 mutations, and in none of cases harboring SLC4A1 mutations. Serum potassium concentration (X ± SD) was higher in SLC4A1 group (3.66 ± 0.44mEq/L) than in ATP6V0A4 group (2.96 ± 0.63mEq/L) (p= 0.046). There were no differences in the other clinical or biochemical variables analyzed in the three groups. This study indicates that non-oriental patients with dRTA caused by mutations in the SLC4A1 gene present later and have normokalemia or milder hypokalemia. Hypoacusia at diagnosis is characteristically associated with ATP6V1B1 gene mutations although it may also be present in infants with ATP6V0A4 defects. Other phenotypical manifestations do not allow predicting the involved gene.

The Molecular Basis for Altered Cation Permeability in Hereditary Stomatocytic Human Red Blood Cells.

Normal human RBCs have a very low basal permeability (leak) to cations, which is continuously corrected by the Na,K-ATPase. The leak is temperature-dependent, and this temperature dependence has been evaluated in the presence of inhibitors to exclude the activity of the Na,K-ATPase and NaK2Cl transporter. The severity of the RBC cation leak is altered in various conditions, most notably the hereditary stomatocytosis group of conditions. Pedigrees within this group have been classified into distinct phenotypes according to various factors, including the severity and temperature-dependence of the cation leak. As recent breakthroughs have provided more information regarding the molecular basis of hereditary stomatocytosis, it has become clear that these phenotypes elegantly segregate with distinct genetic backgrounds. The cryohydrocytosis phenotype, including South-east Asian Ovalocytosis, results from mutations in SLC4A1, and the very rare condition, stomatin-deficient cryohydrocytosis, is caused by mutations in SLC2A1. Mutations in RHAG cause the very leaky condition over-hydrated stomatocytosis, and mutations in ABCB6 result in familial pseudohyperkalemia. All of the above are large multi-spanning membrane proteins and the mutations may either modify the structure of these proteins, resulting in formation of a cation pore, or otherwise disrupt the membrane to allow unregulated cation movement across the membrane. More recently mutations have been found in two RBC cation channels, PIEZO1 and KCNN4, which result in dehydrated stomatocytosis. These mutations alter the activation and deactivation kinetics of these channels, leading to increased opening and allowing greater cation fluxes than in wild type.

Analysis of SLC25A13 gene mutations in five infants with neonatal intrahepatic cholestasis caused by citrin deficiency

OBJECTIVE To identify potential mutations in five infants with neonatal intrahepatic cholestasis caused by citrin deficiency (NICCD). METHODS The SLC25A13 gene was analyzed by next-generation sequencing. Suspected mutations were confirmed by PCR and Sanger sequencing in the probands and their parents. Impact of novel mutations was predicted with PolyPhen-2 software. RESULTS All neonates have harbored mutations of the SLC25A13 gene. Eight mutations were discovered, which included two novel mutations (c.1357A>G and c.1663dup23). All parents were found to be carriers of the mutations. CONCLUSION Mutations of the SLC25A13 gene probably underlie the NICCD among the five patients, among which 851del4 and 1638-1660dup were the most common ones. This has enriched the spectrum of SLC25A13 mutation in association with NICCD.

Genotype–Phenotype Analysis in Pediatric Patients with Distal Renal Tubular Acidosis

Background/Aims: Primary distal renal tubular acidosis (dRTA) in children is a rare genetic disorder, and three causative mutated genes have been identified: SLC4A1, ATP6V1B1, and ATP6V0A4. We analyzed the prevalence and phenotypic differences of genetic mutations in children with dRTA. Methods: A total of 17 children with dRTA were enrolled in the study. All patients underwent genetic testing for all three candidate genes. Results: Pathogenic mutations, including six novel mutations, were detected in 15 (88.2%) patients: dominant SLC4A1 mutations in ten (58.8%) patients, recessive ATP6V0A4 mutations in three (17.6%) patients, and recessive ATP6V1B1 mutations in two (11.8%) patients. Compared to other patients, patients with SLC4A1 mutations showed an older age of onset (3.7 ± 2.6 years) and less severe metabolic acidosis at initial presentation. All patients developed nephrocalcinosis, and sensorineural hearing loss was observed in two patients with ATP6V1B1 mutations. Three (17.6%) patients had decreased renal function (chronic kidney disease stage 2), and five (29.4%) patients had persistent growth retardation at the last follow-up. Long-term prognosis showed no genotype–phenotype correlation. Conclusions: SLC4A1 is the most common defective gene in Korean children with dRTA. Patients with SLC4A1 mutations show later onset and milder disease severity. Long-term follow-up of hearing ability, renal function, and growth is necessary for patients with dRTA.

Two new cases of mitochondrial myopathy with exercise intolerance, hyperlactatemia and cardiomyopathy, caused by recessive SLC25A4 mutations

We report the clinical, morphological and molecular features of two patients with autosomal recessive SLC25A4 (ANT1) gene mutations. Furthermore, all previously published cases are reviewed to identify valuable features for future diagnosis. Patients present a common phenotype with exercise intolerance, hyperlactatemia, and hypertrophic cardiomyopathy. Muscle biopsies show wide sub-sarcolemmal mitochondrial aggregates, and increased activities of all respiratory chain complexes. The phenotype of recessive SLC25A4 (ANT1) mutations although rare, is homogenous and easily recognizable and could help orientate the molecular analysis in adults with exercise intolerance associated with hyperlactatemia.

Molecular diagnosis of citrin deficiency in an infant with intrahepatic cholestasis: identification of a 21.7kb gross deletion that completely silences the transcriptional and translational expression of the affected SLC25A13 allele.

Neonatal Intrahepatic Cholestasis caused by Citrin Deficiency (NICCD) arises from biallelic SLC25A13 mutations, and SLC25A13 analysis provides reliable evidences for NICCD definite diagnosis. However, novel large insertions/deletions in this gene could not be detected just by conventional DNA analysis. This study aimed to explore definite diagnostic evidences for an infant highly-suspected to have NICCD. Prevalent mutation screening and Sanger sequencing of SLC25A13 gene just revealed a paternally-inherited mutation c.851_854del4. Nevertheless, neither citrin protein nor SLC25A13 transcripts of maternal origin could be detected on Western blotting and cDNA cloning analysis, respectively. On this basis, the hidden maternal mutation was precisely positioned using SNP analysis and semi-quantitative PCR, and finally identified as a novel large deletion c.-3251_c.15+18443del21709bp, which involved the SLC25A13 promoter region and the entire exon 1 where locates the translation initiation codon. Hence, NICCD was definitely diagnosed in the infant. To the best of our knowledge, the novel gross deletion, which silenced the transcriptional and translational expression of the affected SLC25A13 allele, is the hitherto largest deletion in SLC25A13 mutation spectrum. The Western blotting approach using mitochondrial protein extracted from expanded peripheral blood lymphocytes, of particular note, might be a new minimally-invasive and more-feasible molecular tool for NICCD diagnosis.

γ-COPI mediates the retention of kAE1 G701D protein in Golgi apparatus - a mechanistic explanation of distal renal tubular acidosis associated with the G701D mutation.

Mutations of the solute carrier family 4 member 1 (SLC4A1) gene encoding kidney anion (chloride/bicarbonate ion) exchanger 1 (kAE1) can cause genetic distal renal tubular acidosis (dRTA). Different SLC4A1 mutations give rise to mutant kAE1 proteins with distinct defects in protein trafficking. The mutant kAE1 protein may be retained in endoplasmic reticulum (ER) or Golgi apparatus, or mis-targeted to the apical membrane, failing to display its function at the baso-lateral membrane. The ER-retained mutant kAE1 interacts with calnexin chaperone protein; disruption of this interaction permits the mutant kAE1 to reach the cell surface and display anion exchange activity. However, the mechanism of Golgi retention of mutant kAE1 G701D protein, which is otherwise functional, is still unclear. In the present study, we show that Golgi retention of kAE1 G701D is due to a stable interaction with the Golgi-resident protein, coat protein complex I (COPI), that plays a role in retrograde vesicular trafficking and Golgi-based quality control. The interaction and co-localization of kAE1 G701D with the γ-COPI subunit were demonstrated in human embryonic kidney (HEK-293T) cells by co-immunoprecipitation and immunofluorescence staining. Small interference RNA (siRNA) silencing of COPI expression in the transfected HEK-293T cells increased the cell surface expression of transgenic kAE1 G701D, as shown by immunofluorescence staining. Our data unveil the molecular mechanism of Golgi retention of kAE1 G701D and suggest that disruption of the COPI-kAE1 G701D interaction could be a therapeutic strategy to treat dRTA caused by this mutant.

Blood glucose and insulin and correlation of SLC25A13 mutations with biochemical changes in NICCD patients.

Neonatal intrahepatic cholestasis caused by citrin deficiency (NICCD) is a hereditary metabolic disease arising from biallelic mutations of SLC25A13. This study aimed to explore the characteristics of fasting blood glucose (FBG), fasting insulin (FINS) and C-peptide (C-P) levels in NICCD infants, analyze their SLC25A13 genetic mutations and further discuss the correlation between SLC25A13 genetic mutations and biochemical changes. Seventy-two cases of infants with cholestasis disease were gathered. Among them, 36 cases with NICCD diagnosis were case group. Meanwhile, 36 cases with unknown etiology but excluded NICCD were control group. FBG, FINS, C-P, ALT, AST, GGT, ALP, TG, HDL-C, LDL-C and Non-HDL-C were collected from all subjects, and DNA was extracted from venous blood for SLC25A13 mutations detection. The incidence of hypoglycemia was 3% in NICCD group. There were no significant statistical difference of FBG, FINS and C-P between NICCD and INC groups ( P > 0.05). ALT, LDL-C and Non-HDL-C levels in NICCD group were lower than the INC group, while SLC25A13 mutations were associated with the level of GGT ( P < 0.05). Ten different SLC25A13 genetic mutations were detected, among which, 851del4, IVS16ins3kb, IVS6+5 G > A and 1638ins23 mutations made up 82% of all mutations. The incidence of hypoglycemia may be higher in small gestational age infants with NICCD. Low LDL-C may be one of the characteristics of dyslipidemia in NICCD infants. There was a correlation between SLC25A13 gene mutations distribution and the GGT level, but the meaning of this finding remains to be further in-depth study. Impact statement This study aims to compare FBG, FINS, C-P, other biochemical and clinical manifestations between NICCD and non-NICCD infants, and discuss differential diagnosis of NICCD and INC beyond the genetic analysis. And investigate the correlation between SLC25A13 genetic mutations and biochemical changes. This work presented that incidence of hypoglycemia may be higher in small gestational age infants with NICCD. Low LDL-C may be one of the characteristics of dyslipidemia in NICCD infants. There was a correlation between SLC25A13 gene mutations distribution and the GGT level.

The genetic and clinical spectrum of a large cohort of patients with distal renal tubular acidosis

Primary distal renal tubular acidosis is a rare genetic disease. Mutations in SLC4A1, ATP6V0A4, and ATP6V1B1 genes have been described as the cause of the disease, transmitted as either an autosomal dominant or recessive trait. Particular clinical features, such as sensorineural hearing loss, have been mainly described in association with mutations in one gene instead of the others. Nevertheless, the diagnosis of distal renal tubular acidosis is essentially based on clinical and laboratory findings, and the series of patients described so far are usually represented by small cohorts. Therefore, a strict genotype-phenotype correlation is still lacking, and questions about whether clinical and laboratory data should direct the genetic analysis remain open. Here, we applied next-generation sequencing in 89 patients with a clinical diagnosis of distal renal tubular acidosis, analyzing the prevalence of genetic defects in SLC4A1, ATP6V0A4, and ATP6V1B1 genes and the clinical phenotype. A genetic cause was determined in 71.9% of cases. In our group of sporadic cases, clinical features, including sensorineural hearing loss, are not specific indicators of the causal underlying gene. Mutations in the ATP6V0A4 gene are quite as frequent as mutations in ATP6V1B1 in patients with recessive disease. Chronic kidney disease was frequent in patients with a long history of the disease. Thus, our results suggest that when distal renal tubular acidosis is suspected, complete genetic testing could be considered, irrespective of the clinical phenotype of the patient.

Value of albumin in diagnosis of neonatal intrahepatic cholestasis caused by citrin deficiency

Objective: To investigate the clinical value of albumin (Alb) in the diagnosis of neonatal intrahepatic cholestasis caused by citrin deficiency (NICCD). Methods: A retrospective analysis was performed for the clinical data of 90 children with NICCD who visited Children's Hospital of Fudan University from January 2007 to December 2014, and according to the content of Alb, these children were divided into Alb < 30 g/L (LA) group with 20 children and Alb ≥30 g/L (NA) group with 70 children. The clinical manifestations, results of laboratory examination, results of blood tandem mass spectrometry and urine gas chromatography-mass spectrometry, and gene detection results were compared between the two groups. The t-test and the chi-square test were used for statistical analysis.. Results: There were significant differences between the LA group and the NA group in splenomegaly degree (3.28±1.95 cm vs 1.92±1.06 cm, P = 0.030), aspartate aminotransferase/alanine aminotransferase ratio [3.15 (0.38-5.93) vs 2.14 (0.26-6.67), P = 0.010], activated partial thromboplastin time (53.27±11.68 s vs 45.06±9.79 s, P = 0.003), and international normalized ratio (1.92±1.35 vs 1.29±0.33, P = 0.001). The SLC25A13 mutation I 851_854del4 was associated with Alb (χ2 = 4.76, P = 0.025). Conclusion: As for the children with Alb < 30g/L who are highly suspected of having NICCD, SLC25A13 gene detection and blood/urine mass spectrometry should be performed as early as possible, in order to initiate intervention treatment as soon as possible, prevent and treat complications, and improve prognosis.

Recurrent De Novo Dominant Mutations in SLC25A4 Cause Severe Early-Onset Mitochondrial Disease and Loss of Mitochondrial DNA Copy Number

Mutations in SLC25A4 encoding the mitochondrial ADP/ATP carrier AAC1 are well-recognized causes of mitochondrial disease. Several heterozygous SLC25A4 mutations cause adult-onset autosomal-dominant progressive external ophthalmoplegia associated with multiple mitochondrial DNA deletions, whereas recessive SLC25A4 mutations cause childhood-onset mitochondrial myopathy and cardiomyopathy. Here, we describe the identification by whole-exome sequencing of seven probands harboring dominant, de novo SLC25A4 mutations. All affected individuals presented at birth, were ventilator dependent and, where tested, revealed severe combined mitochondrial respiratory chain deficiencies associated with a marked loss of mitochondrial DNA copy number in skeletal muscle. Strikingly, an identical c.239G>A (p.Arg80His) mutation was present in four of the seven subjects, and the other three case subjects harbored the same c.703C>G (p.Arg235Gly) mutation. Analysis of skeletal muscle revealed a marked decrease of AAC1 protein levels and loss of respiratory chain complexes containing mitochondrial DNA-encoded subunits. We show that both recombinant AAC1 mutant proteins are severely impaired in ADP/ATP transport, affecting most likely the substrate binding and mechanics of the carrier, respectively. This highly reduced capacity for transport probably affects mitochondrial DNA maintenance and in turn respiration, causing a severe energy crisis. The confirmation of the pathogenicity of these de novo SLC25A4 mutations highlights a third distinct clinical phenotype associated with mutation of this gene and demonstrates that early-onset mitochondrial disease can be caused by recurrent de novo mutations, which has significant implications for the application and analysis of whole-exome sequencing data in mitochondrial disease.

P.297 - Two new cases of mitochondrial myopathy with exercise intolerance, lactic acidosis and cardiomyopathy, caused by recessive SLC25A4 mutations

P.297 - Two new cases of mitochondrial myopathy with exercise intolerance, lactic acidosis and cardiomyopathy, caused by recessive SLC25A4 mutations

Clinical analysis and follow-up of neonatal intrahepatic cholestasis caused by Citrin deficiency in 29 children

Objective To analyze the main clinical manifestations, laboratory features and prognosis of neonatal intrahepatic cholestasis caused by Citrin defiency (NICCD). Methods Twenty-nine NICCD infants were diagnosed by blood tandem mass spectrometry (MS-MS)analysis and/or SLC25A13 mutation analysis from July 2012 to February 2015 in Children's Hospital of Chongqing Medical University.Clinical data of 29 cases were analyzed retrospectively which included manifestations, laboratory features and prognosis.The general situation, feeding, liver function, growth were followed up. Results Twenty-nine infants suffering from NICCD presented jaundice in an early time, and some clinical manifestations were investigated such as hepatomegaly (20/29 cases), splenomegaly (3/29 cases), anemia (14/29 cases), and failure to thrive (9/29 cases). Laboratory data suggested that all of 29 patients had increased conjugated bilirubin, total bile acid, γ-glutamyl transferase and alkaline phosphatase. Some patients also showed abnormal coagulation function (20/22 cases), dyslipidemia (9/20 cases), increased blood lactic acid (22/26 cases) and alpha-fetoprotein (14/14 cases), decreased albumin(24/29 cases), blood glucose(17/22 cases) and ceruloplasmin(4/4 cases). The pathological analysis of one patient's liver indicated the edema and degeneration of liver cells, intrahepatic cholestasis and a small amount of fibrous tissue hyperplasia in portal area.MS-MS analysis of blood samples revealed distinctive increase in methionine, tyrosine, threonine, citrulline, arginine and free carnitine, long-chain acyl-carnitine in most patients.Gas chromatography-mass spectrometer (GC-MS) analysis of urine samples mainly showed elevated 4-hydroxyphenyllactic acid and 4-hydroxyphenylpyruvic acid.Prognosis showed that most of the NICCD patients (8/29 cases) could recover in one-year old with a lactose-free, medium chain triglyceride-enriched formula, and one patient died of liver cirrhosis.Three patients at over one-year old had the preference of a high protein and low carbohydrate diet. Conclusions Infants might be considered to have NICCD if they have jaundice in an early time, with the clinical characteristics of hepatomegaly, splenomegaly, abnormal coagulation function, anemia, failure to thrive, dyslipidemia, decresed albumin and blood glucose, increased blood lactic acid and alpha-fetoprotein.After that further tests of MS-MS, GC-MS and gene analysis of this disease are needed to confirm diagnosis. Key words: Citrin deficiency; Intrahepatic cholestasis; Clinical manifestation; Laboratory finding; Prognosis

Absence of Band 3 in Severe Dyserythropoietic/Hemolytic Anemia with Complete Distal Renal Acidosis and a Novel Homozygous Exon 12 C.1430C>a (p.Ser477X) SLC4A1 Gene Mutation

The solute carrier 4A1 gene (SLC4A1) encodes theband 3 or bicarbonate anionic exchanger 1 (AE1). It is not only the major glycoprotein of the red blood cell (RBC) membrane but also expressed in acid secreting alpha-intercalated kidney cells. Functionally impairing SLC4A1 mutations reduce the expression and/or activity of AE1 thereby causing a unique combination of hemolytic anemia and distal renal tubular acidosis (dRTA). So far, only four such particular homozygote mutations have been documented in humans: an exon 11 p.400-408 deletion in Southeast Asian ovalocytosis (SAO) with transfusion-dependent dyserythropoietic anemia and dRTA (Picard et al, Blood 123:1963;2014), an exon 13 p.V488M mutation in transfusion-dependent hereditary spherocytosis (HS) and dRTA lacking band 3 (Ribeiro et al, Blood 96:1602;2000), an exon 16 p.S667F mutation in transfusion-dependent HS and incomplete dRTA (Toye et al, Blood 111:5380;2008), and finally an exon 19 p.Ala858Asp in a compensated hemolytic anemia with marked acanthocytosis, echinocytosis and dRTA (Fawaz et al, Europ J Haematol 88:350;2012). We report herein a novel homozygote variant in exon 12 in a patient with a transfusion-dependent dyserythropoietic/hemolytic anemia and complete dRTA.The now 4-years old Turkish boy was born after 32 weeks of gestation and presented with a severe hemolytic anemia (Hb 40 g/L) that required exchange transfusions and a complete dRTA that was treated with oral bicarbonate. He also suffered from delayed psychomotoric developmental with failure to thrive, trigonocephalus and strabismus convergens. Bone marrow smears showed marked dyserythropoiesis but normal myeloid and megakaryocytic lineages. Although necessary monthly transfusions impeded the patient's direct diagnostic work-up, a flow cytometric eosin-5-maleimide assay eventually revealed a reduced staining of his consanguine parents' and his two siblings' erythrocytes, who all had subclinical signs of spherocytosis despite normal RBC counts.Based on these findings, we analyzed the SLC4A1 gene and found two homozygous sequence variants in the patient, namely a novel disease-relevant exon 12 nonsense mutation c.1430C>A (p.Ser477X) and a disease-unrelated c.2312-48T>G (rs13306780). The ensuing stop codon of the former truncates the protein, prevents band 3 formation and reduces glycophorin A expression. Bright field imaging uncovered few phenotypic spherocytic band 3 null RBCs even in the peripheral blood.In accordance with the autosomal recessive inheritance pattern, both healthy parents as well as his healthy siblings were found to be heterozygous carriers. The band 3 protein was reduced to 50-60% in the parents' erythrocytes. An increased approximately 22 kDa-sized band was evident in Coomassie stained gels of the heterozygous mutation carriers' membrane preparations and classified by immunoblotting as peroxiredoxin 2 (PRDX2), which plays a major role in protecting RBCs from oxidative stress.Taken together, the provided data clearly confirm the relevance of this particular c.1430C>A (p.Ser477X) SLC4A1 mutation in the disease process. Of note, such a severe dyserythropoietic anemia and complete dRTA was also recently reported in a patient with Southeast Asian ovalocytosis and another form of homozygous SLC4A1 mutation (Picard et al, Blood 123:1963;2014). DisclosuresNo relevant conflicts of interest to declare.

Abnormal Newborn Screening in a Healthy Infant of a Mother with Undiagnosed Medium-Chain Acyl-CoA Dehydrogenase Deficiency.

A neonate with low blood free carnitine level on newborn tandem mass spectrometry screening was evaluated for possible carnitine transporter defect (CTD). The plasma concentration of free carnitine was marginally reduced, and the concentrations of acylcarnitines (including C6, C8, and C10:1) were normal on confirmatory tests. Organic acids in urine were normal. In addition, none of the frequent Faroese SLC22A5 mutations (p.N32S, c.825-52G>A) which are common in the Danish population were identified. Evaluation of the mother showed low-normal free carnitine, but highly elevated medium-chain acylcarnitines (C6, C8, and C10:1) consistent with medium-chain acyl-CoA dehydrogenase deficiency (MCADD). The diagnosis was confirmed by the finding of homozygous presence of the c.985A>G mutation in ACADM.

Clinical, Molecular and Functional Investigation on an Infant with Neonatal Intrahepatic Cholestasis Caused by Citrin Deficiency (NICCD)

Background and Objective SLC25A13 analysis has provided reliable evidences for the definitive diagnosis of citrin deficiency (CD) in the past decade. Meanwhile, these studies generated some issues yet to be resolved, including the pathogenicity of SLC25A13 missense mutations and the mRNA product from the mutation c.615+5G>A. This study aims to investigate the effect of a novel missense mutation on the aspartate/glutamate carrier (AGC) function of citrin protein, and to explore the aberrant transcript from c.615+5G>A in the same CD infant.Methods and ResultsBy means of screening for prevalent SLC25A13 mutations and exons sequencing, the patient proved a compound heterozygote of c.615+5G>A and a novel c.1064G>A (p.Arg355Gln) mutation. An aberrant transcript with retention of the entire intron 6, r.[615+1_615+1789ins; 615+5 g>a] (GenBank accession number KJ128074), which was resulted from c.615+5G>A, was detected by RT-PCR and cDNA sequencing. After bioinformatic analyses of the novel missense mutation c.1064G>A, the growth abilities of three agc1Δ yeast strains were tested, which had been transformed with recombinant or empty vectors, respectively. Besides the bioinformatically pathogenic evidences, the growth ability of the agc1Δ strains transformed with mutant recombinant was the same as with empty vector, but significantly lower than that with normal control in functional analysis.ConclusionsA CD infant was definitely diagnosed in this paper by a genetic, transcriptional and functional analysis of SLC25A13 gene. This study provided direct laboratory evidences supporting the splice-site nature of the c.615+5G>A mutation, and the novel c.1064G>A variation, which proved a pathogenic mutation bioinformatically and functionally, enriched the SLC25A13 mutation spectrum.

Screening for five prevalent mutations of SLC25A13 gene in Guangdong, China: a molecular epidemiologic survey of citrin deficiency.

Citrin is the liver-type aspartate/glutamate carrier isoform 2 (AGC2) encoded by SLC25A13 gene, playing important roles in the urea cycle and the malate-aspartate shuttle. Citrin deficiency (CD) has proven a disease entity with high prevalence in south China, including Guangdong with the largest population, but CD epidemiology in this province has not been well characterized. This study aims to screen for five prevalent SLC25A13 mutations, c.851_854del (p.R284fs286X), c.1638_1660dup (p.A554fs570X), c.615+5G>A (p.A206fs212X), IVS16ins3kb (p.A584fs585X) and c.1399C>T (p.R467X), to calculate the mutation carrier rate in Guangdong. A total of 2,428 used blood samples for health examination were collected as the research subjects, including 1,558 from 5 cities in the Pearl River Delta area and the remaining 870 from 4 peripheral cities, and the 5 mutations screened using High Resolution Melting Assay and HybProbe assay. A total of 52 carriers were detected, including 2 carriers of a novel c.1420G>A (p.V474M) mutation that impairs citrin function, as judged by the functional analysis in the yeast system. The carrier rate was higher in Pearl River Delta area than that in the peripheral cities (26/1,558 vs. 26/870, with χ(2) = 4.639 and P < 0.05). The carrier rate was around 1/47 (52/2,428), theoretically with the CD morbidity of 1/8,800 and the number of CD patients over 11,800 in Guangdong population. This study has provided primary epidemiologic data for the evaluation of CD effect in Guangdong province. Moreover, the newly identified c.1420G>A mutation that impairs AGC2 function has enriched the mutation spectrum of human SLC25A13 gene.