Biallelic NDC1 variants that interfere with ALADIN binding are associated with neuropathy and Triple-A-like syndrome.

Nuclear pore complexes (NPC) regulate nucleocytoplasmic transport and are anchored in the nuclear envelope by the transmembrane nucleoporin NDC1. NDC1 is essential for post-mitotic NPC assembly and the recruitment of ALADIN to the nuclear envelope. While no human disorder has been associated to one of the three transmembrane nucleoporins, biallelic variants in AAAS, encoding ALADIN, cause Triple-A syndrome (Allgrove syndrome). Triple A syndrome, characterized by alacrima, achalasia and adrenal insufficiency, often includes progressive demyelinating polyneuropathy and other neurological complaints. In this report, diagnostic exome and/or RNA-sequencing was performed in 7 individuals from 4 unrelated consanguineous families with AAAS negative triple A syndrome. Molecular and clinical studies followed to elucidate the pathogenic mechanism. The affected individuals presented with intellectual disability, motor impairment, severe demyelinating with secondary axonal polyneuropathy, alacrima and achalasia.. None of the affected individuals has adrenal insufficiency. All individuals presented with biallelic NDC1 in-frame deletions or missense variants, that affect amino acids and protein domains required for ALADIN binding. No other significant variants associated with the phenotypic features were reported. Skin fibroblasts derived from affected individuals show decreased recruitment of ALADIN to the NE and decreased post-mitotic NPC insertion, confirming pathogenicity of the variants. Taken together, our results implicate biallelic NDC1 variants in the pathogenesis of polyneuropathy and a Triple-A-like disorder without adrenal insufficiency, by interfering with physiological NDC1 functions, including the recruitment of ALADIN to the NPC.

A Humanized and Viable Animal Model for Congenital Adrenal Hyperplasia-CYP21A2-R484Q Mutant Mouse.

Congenital Adrenal Hyperplasia (CAH) is an autosomal recessive disorder impairing cortisol synthesis due to reduced enzymatic activity. This leads to persistent adrenocortical overstimulation and the accumulation of precursors before the blocked enzymatic step. The predominant form of CAH arises from mutations in CYP21A2, causing 21-hydroxylase deficiency (21-OHD). Despite emerging treatment options for CAH, it is not always possible to physiologically replace cortisol levels and counteract hyperandrogenism. Moreover, there is a notable absence of an effective in vivo model for pre-clinical testing. In this work, we developed an animal model for CAH with the clinically relevant point mutation p.R484Q in the previously humanized CYP21A2 mouse strain. Mutant mice showed hyperplastic adrenals and exhibited reduced levels of corticosterone and 11-deoxycorticosterone and an increase in progesterone. Female mutants presented with higher aldosterone concentrations, but blood pressure remained similar between wildtype and mutant mice in both sexes. Male mutant mice have normal fertility with a typical testicular appearance, whereas female mutants are infertile, exhibit an abnormal ovarian structure, and remain in a consistent diestrus phase. Conclusively, we show that the animal model has the potential to contribute to testing new treatment options and to prevent comorbidities that result from hormone-related derangements and treatment-related side effects in CAH patients.

Fertility and sexual activity in patients with Triple A syndrome.

Objective: Triple A syndrome, caused by autosomal recessively inherited mutations in the AAAS gene is characterized by alacrima, achalasia, adrenal insufficiency, and neurological impairment. To the best of our knowledge, no patients of both sexes have been reported to have offspring. Our aim was to assess the causes of infertility in male patients with this multisystemic syndrome, and to present a female patient that spontaneously conceived a child. Design: Cross-sectional study. Methods: Six males aged 19-48 years were included. Gonadotropins, testosterone, DHEAS, androstenedione, inhibin B, anti-Mullerian hormone measurements and testicular ultrasound were performed. Results: All six male patients had impaired general health and neurological symptoms including erectile and ejaculatory dysfunction. None of them had an offspring. The only demonstrated cause of infertility in our male patients was erectile and ejaculatory dysfunction which precludes sexual intercourse. Our patients had normal libido but were sexually abstinent. Except for low adrenal androgen levels, the concentrations of all measured hormones as well as testicular ultrasound were normal which may indicate the possibility of spermatogenesis in male patients with triple A syndrome. Little is known about fertility in female patients, but based on our observations spontaneous pregnancies seem to be possible. Conclusion: Our results contribute to still scarce knowledge on fertility in patients with Triple A syndrome and as well represents a foundation for further research on causes of infertility and possible treatment options.

Acromicric dysplasia due to a novel missense mutation in the fibrillin 1 gene in a three-generation family.

OBJECTIVES: Short stature is one of the most common reasons for consulting a paediatric endocrinologist. Targeted diagnosis of familial short stature can be challenging due to a broad spectrum of differential diagnoses. CASE PRESENTATION: Here we report a novel mutation in the fibrillin 1 gene (FBN1) in six family members causing a mild phenotype of acromicric dysplasia. Additionally, we present the effects of growth hormone therapy in one of the affected children. CONCLUSIONS: Acromicric dysplasia is a very rare skeletal dysplasia with a prevalence of <1 of 1.000.000 with only about 60 cases being reported worldwide. It is characterized by short stature, acromelia, mild facial dysmorphy but normal intelligence. This study aims to exemplify the clinical and molecular features of FBN1-related acromicric dysplasia and illustrates its pleiotropy by presenting a new, mild phenotype.

CYP21A2 Gene Expression in a Humanized 21-Hydroxylase Mouse Model Does Not Affect Adrenocortical Morphology and Function.

Steroid 21-hydroxylase is an enzyme of the steroid pathway that is involved in the biosynthesis of cortisol and aldosterone by hydroxylation of 17α-hydroxyprogesterone and progesterone at the C21 position. Mutations in CYP21A2, the gene encoding 21-hydroxylase, cause the most frequent form of the autosomal recessive disorder congenital adrenal hyperplasia (CAH). In this study, we generated a humanized 21-hydroxylase mouse model as the first step to the generation of mutant mice with different CAH-causing mutations. We replaced the mouse Cyp21a1 gene with the human CYP21A2 gene using homologous recombination in combination with CRISPR/Cas9 technique. The aim of this study was to characterize the new humanized mouse model. All results described are related to the homozygous animals in comparison with wild-type mice. We show analogous expression patterns of human 21-hydroxylase by the murine promoter and regulatory elements in comparison to murine 21-hydroxylase in wild-type animals. As expected, no Cyp21a1 transcript was detected in homozygous CYP21A2 adrenal glands. Alterations in adrenal gene expression were observed for Cyp11a1, Star, and Cyb11b1. These differences, however, were not pathological. Outward appearance, viability, growth, and fertility were not affected in the humanized CYP21A2 mice. Plasma steroid levels of corticosterone and aldosterone showed no pathological reduction. In addition, adrenal gland morphology and zonation were similar in both the humanized and the wild-type mice. In conclusion, humanized homozygous CYP21A2 mice developed normally and showed no differences in histological analyses, no reduction in adrenal and gonadal gene expression, or in plasma steroids in comparison with wild-type littermates.

Genetic Analysis of Pediatric Primary Adrenal Insufficiency of Unknown Etiology: 25 Years' Experience in the UK.

CONTEXT: Although primary adrenal insufficiency (PAI) in children and young people is often due to congenital adrenal hyperplasia (CAH) or autoimmunity, other genetic causes occur. The relative prevalence of these conditions is poorly understood. OBJECTIVE: We investigated genetic causes of PAI in children and young people over a 25 year period. DESIGN SETTING AND PARTICIPANTS: Unpublished and published data were reviewed for 155 young people in the United Kingdom who underwent genetic analysis for PAI of unknown etiology in three major research centers between 1993 and 2018. We pre-excluded those with CAH, autoimmune, or metabolic causes. We obtained additional data from NR0B1 (DAX-1) clinical testing centers. INTERVENTION AND OUTCOME MEASUREMENTS: Genetic analysis involved a candidate gene approach (1993 onward) or next generation sequencing (NGS; targeted panels, exomes) (2013-2018). RESULTS: A genetic diagnosis was reached in 103/155 (66.5%) individuals. In 5 children the adrenal insufficiency resolved and no genetic cause was found. Pathogenic variants occurred in 11 genes: MC2R (adrenocorticotropin receptor; 30/155, 19.4%), NR0B1 (DAX-1; 7.7%), CYP11A1 (7.7%), AAAS (7.1%), NNT (6.5%), MRAP (4.5%), TXNRD2 (4.5%), STAR (3.9%), SAMD9 (3.2%), CDKN1C (1.3%), and NR5A1/steroidogenic factor-1 (SF-1; 0.6%). Additionally, 51 boys had NR0B1 variants identified through clinical testing. Although age at presentation, treatment, ancestral background, and birthweight can provide diagnostic clues, genetic testing was often needed to define the cause. CONCLUSIONS: PAI in children and young people often has a genetic basis. Establishing the specific etiology can influence management of this lifelong condition. NGS approaches improve the diagnostic yield when many potential candidate genes are involved.

Homozygous mutation in murine retrovirus integration site 1 gene associated with a non-syndromic form of isolated familial achalasia.

BACKGROUND: Achalasia is a condition characterized by impaired function of esophageal motility and incomplete relaxation of the lower esophagus sphincter, causing dysphagia and regurgitation. Rare cases of early-onset achalasia appear often in combination with further symptoms in a syndromic form as an inherited disease. METHODS: Whole genome sequencing was used to investigate the genetic basis of isolated achalasia in a family of Tunisian origin. We analyzed the function of the affected protein with immunofluorescence and affinity chromatography study. KEY RESULTS: A homozygous nonsense mutation was detected in murine retrovirus integration site 1 (MRVI1) gene (Human Genome Organisation Gene Nomenclature Committee (HGNC) approved gene symbol: IRAG1) encoding the inositol 1,4,5-trisphosphate receptor 1 (IP3 R1)-associated cyclic guanosine monophosphate (cGMP) kinase substrate (IRAG). Sanger sequencing confirmed co-segregation of the mutation with the disease. Sequencing of the entire MRVI1 gene in 35 additional patients with a syndromic form of achalasia did not uncover further cases with MRVI1 mutations. Immunofluorescence analysis of transfected COS7 cells revealed GFP-IRAG with the truncating mutation p.Arg112* (transcript variant 1) or p.Arg121* (transcript variant 2) to be mislocalized in the cytoplasm and the nucleus. Co-transfection with cGMP-dependent protein kinase 1 isoform ß (cGK1ß) depicted a partial mislocalization of cGK1ß due to mislocalized truncated IRAG. Isolation of protein complexes revealed that the truncation of this protein causes the loss of the interaction domain of IRAG with cGK1ß. CONCLUSIONS & INFERENCES: In individuals with an early onset of achalasia without further accompanying symptoms, MRVI1 mutations should be considered as the disease-causing defect.

A novel homozygous nonsense mutation of VPS13B associated with previously unreported features of Cohen syndrome.

Cohen syndrome (CS) is a rare autosomal recessive disorder associated with mutations in the vacuolar protein sorting 13 homolog B (VPS13B; formerly COH1) gene. The core clinical phenotype comprises a characteristic facial gestalt, marked developmental delay, and myopia. Additional, nonobligatory features include obesity, microcephaly, short stature, muscular hypotonia, scoliosis, narrow hands and feet, progressive retinopathy, as well as neutropenia. Here we report a novel homozygous nonsense mutation in the VPS13B gene and previously undescribed clinical features in a 19-year-old woman with developmental delay, intellectual disability, and a particular facial appearance. The patient showed several features consistent with CS. In addition, the parents observed congenital alacrima and anhidrosis persisting until onset of puberty. The diagnosis was not established based on the clinical phenotype. We performed whole-genome sequencing and identified a novel homozygous nonsense mutation c.62T>G (NM_152564.4), p.(Leu21*) in the VPS13B gene. Our findings extended the previously reported phenotype of CS. We conclude that transient, prepubertal alacrima and anhidrosis are part of the phenotypic spectrum of CS associated with a novel homozygous nonsense mutation in the VPS13B gene.

Homozygous deletion of the entire AAAS gene in a triple A syndrome patient.

Triple A syndrome, a multisystemic autosomal recessive disease, is characterized by the clinical triad of adrenal insufficiency, alacrima and achalasia in combination with progressive neurological impairments. The disorder is caused by homozygous or compound heterozygous mutations in the AAAS gene. Here we present the clinical and molecular data of a ten year old patient with triple A syndrome. Array CGH analysis confirmed the PCR-based assumption of a homozygous deletion of the entire AAAS gene in the patient and a heterozygous deletion in both parents. We demonstrate that the patient carries a 15 kb deletion and identified the 5' and 3' breakpoints outside the AAAS gene. This is the first report of a triple A syndrome patient with a homozygous deletion of the entire AAAS gene.

Modeling the Pathological Long-Range Regulatory Effects of Human Structural Variation with Patient-Specific hiPSCs.

The pathological consequences of structural variants disrupting 3D genome organization can be difficult to elucidate in vivo due to differences in gene dosage sensitivity between mice and humans. This is illustrated by branchiooculofacial syndrome (BOFS), a rare congenital disorder caused by heterozygous mutations within TFAP2A, a neural crest regulator for which humans, but not mice, are haploinsufficient. Here, we present a BOFS patient carrying a heterozygous inversion with one breakpoint located within a topologically associating domain (TAD) containing enhancers essential for TFAP2A expression in human neural crest cells (hNCCs). Using patient-specific hiPSCs, we show that, although the inversion shuffles the TFAP2A hNCC enhancers with novel genes within the same TAD, this does not result in enhancer adoption. Instead, the inversion disconnects one TFAP2A allele from its cognate enhancers, leading to monoallelic and haploinsufficient TFAP2A expression in patient hNCCs. Our work illustrates the power of hiPSC differentiation to unveil long-range pathomechanisms.

Triple A patient cells suffering from mitotic defects fail to localize PGRMC1 to mitotic kinetochore fibers.

BACKGROUND: Membrane-associated progesterone receptors are restricted to the endoplasmic reticulum and are shown to regulate the activity of cytochrome P450 enzymes which are involved in steroidogenesis or drug detoxification. PGRMC1 and PGRMC2 belong to the membrane-associated progesterone receptor family and are of interest due to their suspected role during cell cycle. PGRMC1 and PGRMC2 are thought to bind to each other; thereby suppressing entry into mitosis. We could previously report that PGRMC2 interacts with the nucleoporin ALADIN which when mutated results in the autosomal recessive disorder triple A syndrome. ALADIN is a novel regulator of mitotic controller Aurora kinase A and depletion of this nucleoporin leads to microtubule instability. RESULTS: In the current study, we present that proliferation is decreased when ALADIN, PGRMC1 or PGRMC2 are over-expressed. Furthermore, we find that depletion of ALADIN results in mislocalization of Aurora kinase A and PGRMC1 in metaphase cells. Additionally, PGRMC2 is over-expressed in triple A patient fibroblasts. CONCLUSION: Our results emphasize the possibility that loss of the regulatory association between ALADIN and PGRMC2 gives rise to a depletion of PGRMC1 at kinetochore fibers. This observation may explain part of the symptoms seen in triple A syndrome patients.

"Crying without tears" as an early diagnostic sign-post of triple A (Allgrove) syndrome: two case reports.

BACKGROUND: Triple A syndrome (or Allgrove syndrome) is a rare autosomal recessive disorder characterized by alacrima, achalasia, adrenal insufficiency and autonomic/neurological abnormalities. The majority of cases are caused by mutations in the AAAS gene located on chromosome 12q13. However, the clinical picture as well as genetic testing may be complex since symptomatology is variable and mutations cannot be identified in all clinically diagnosed patients. We present two unrelated patients with triple-A syndrome illustrating the importance of alacrima as an early clinical sign. CASE PRESENTATION: A 3.5 year old girl presented with repeated hypoglycaemic myoclonic events. Adrenal insufficiency was diagnosed. In addition, alacrima, obvious since early infancy, was incidentally reported by the mother and finally lead to the clinical diagnosis of triple A syndrome. This was confirmed by positive mutation analysis of the AAAS gene. The second patient, an 8 months old boy was presented because of anisocoria and unilateral optic atrophy. MRI revealed cerebellar vermis hypotrophy. Psychomotor retardation, failure to thrive, and frequent vomiting lead to further diagnostic work-up. Achalasia was diagnosed radiologically. In addition, the mother mentioned absence of tears since birth leading to the clinical diagnosis of triple A syndrome. In contrast to the first cases genetic testing was negative. CONCLUSION: These two patients illustrate the heterogeneity of triple A syndrome in both terms, clinical expression and genetic testing. We particularly aim to stress the importance of alacrima, which should be considered as a red flag symptom. Further differential diagnosis is required in every child affected by alacrima.

Compensation for chronic oxidative stress in ALADIN null mice.

Mutations in the AAAS gene coding for the nuclear pore complex protein ALADIN lead to the autosomal recessive disorder triple A syndrome. Triple A patients present with a characteristic phenotype including alacrima, achalasia and adrenal insufficiency. Patient fibroblasts show increased levels of oxidative stress, and several in vitro studies have demonstrated that the nucleoporin ALADIN is involved in both the cellular oxidative stress response and adrenal steroidogenesis. It is known that ALADIN knock-out mice lack a phenotype resembling human triple A syndrome. The objective of this study was to determine whether the application of chronic oxidative stress by ingestion of paraquat would generate a triple A-like phenotype in ALADIN null mice. Adult male mice were fed either a paraquat (0.25 g/kg diet) or control diet for 11 days. After application of chronic oxidative stress, ALADIN knock-out mice presented with an unexpected compensated glutathione metabolism, but lacked a phenotype resembling human triple A syndrome. We did not observe increased levels of oxidative stress and alterations in adrenal steroidogenesis in mice depleted for ALADIN. This study stresses the species-specific role of the nucleoporin ALADIN, which in mice involves a novel compensatory mechanism for regulating the cellular glutathione redox response.

Two patients with MIRAGE syndrome lacking haematological features: role of somatic second-site reversion SAMD9 mutations.

BACKGROUND: Myelodysplasia, infection, restriction of growth, adrenal hypoplasia, genital phenotypes and enteropathy (MIRAGE) syndrome is a recently described congenital disorder caused by heterozygous SAMD9 mutations. The phenotypic spectrum of the syndrome remains to be elucidated. METHODS AND RESULTS: We describe two unrelated patients who showed manifestations compatible with MIRAGE syndrome, with the exception of haematological features. Leucocyte genomic DNA samples were analysed with next-generation sequencing and Sanger sequencing, revealing the patients to have two de novoSAMD9 mutations on the same allele (patient 1 p.[Gln695*; Ala722Glu] and patient 2 p.[Gln39*; Asp769Gly]). In patient 1, p.Gln695* was absent in genomic DNA extracted from hair follicles, implying that the non-sense mutation was acquired somatically. In patient 2, with the 46,XX karyotype, skewed X chromosome inactivation pattern was found in leucocyte DNA, suggesting monoclonality of cells in the haematopoietic system. In vitro expression experiments confirmed the growth-restricting capacity of the two missense mutant SAMD9 proteins that is a characteristic of MIRAGE-associated SAMD9 mutations. CONCLUSIONS: Acquisition of a somatic nonsense SAMD9 mutation in the cells of the haematopoietic system might revert the cellular growth repression caused by the germline SAMD9 mutations (ie, second-site reversion mutations). Unexpected lack of haematological features in the two patients would be explained by the reversion mutations.

Phenotype-genotype spectrum of AAA syndrome from Western India and systematic review of literature.

OBJECTIVE: To study genotype-phenotype spectrum of triple A syndrome (TAS). METHODS: Retrospective chart analysis of Indian TAS patients (cohort 1, n = 8) and review of genotyped TAS cases reported in world literature (cohort 2, n = 133, 68 publications). RESULTS: Median age at presentation was 4.75 years (range: 4-10) and 5 years (range: 1-42) for cohorts 1 and 2, respectively. Alacrima, adrenal insufficiency (AI), achalasia and neurological dysfunction (ND) were seen in 8/8, 8/8, 7/8 and 4/8 patients in cohort 1, and in 99, 91, 93 and 79% patients in cohort 2, respectively. In both cohorts, alacrima was present since birth while AI and achalasia manifested before ND. Mineralocorticoid deficiency (MC) was uncommon (absent in cohort 1, 12.5% in cohort 2). In cohort 1, splice-site mutation in exon 1 (p.G14Vfs*45) was commonest, followed by a deletion in exon 8 (p.S255Vfs*36). Out of 65 mutations in cohort 2, 14 were recurrent and five exhibited regional clustering. AI was more prevalent, more often a presenting feature, and was diagnosed at younger age in T group (those with truncating mutations) as compared to NT (non-truncating mutations) group. ND was more prevalent, more common a presenting feature, with later age at onset in NT as compared to T group. CONCLUSION: Clinical profile of our patients is similar to that of patients worldwide. Alacrima is the earliest and most consistent finding. MC deficiency is uncommon. Some recurrent mutations show regional clustering. p.G14Vfs*45 and p.S255Vfs*36 account for majority of AAAS mutations in our cohort. Phenotype of T group differs from that of NT group and merits future research.

ALADIN is required for the production of fertile mouse oocytes.

Asymmetric cell divisions depend on the precise placement of the spindle apparatus. In mammalian oocytes, spindles assemble close to the cell's center, but chromosome segregation takes place at the cell periphery where half of the chromosomes are expelled into small, nondeveloping polar bodies at anaphase. By dividing so asymmetrically, most of the cytoplasmic content within the oocyte is preserved, which is critical for successful fertilization and early development. Recently we determined that the nucleoporin ALADIN participates in spindle assembly in somatic cells, and we have also shown that female mice homozygously null for ALADIN are sterile. In this study we show that this protein is involved in specific meiotic stages, including meiotic resumption, spindle assembly, and spindle positioning. In the absence of ALADIN, polar body extrusion is compromised due to problems in spindle orientation and anchoring at the first meiotic anaphase. ALADIN null oocytes that mature far enough to be fertilized in vitro are unable to support embryonic development beyond the two-cell stage. Overall, we find that ALADIN is critical for oocyte maturation and appears to be far more essential for this process than for somatic cell divisions.

Triple A Syndrome: Preliminary Response to the Antioxidant N-Acetylcysteine Treatment in a Child.

INTRODUCTION: Triple A syndrome (AAAS) is a rare autosomal recessive disorder characterized by alacrima, achalasia, ACTH-resistant adrenal insufficiency, autonomic dysfunction, and progressive neurodegeneration. Increased oxidative stress, demonstrated in patients' fibroblasts in vitro, may be a central disease mechanism. N-acetylcysteine protects renal function in patients with kidney injuries associated with increased oxidative stress and improves viability of AAAS-knockdown adrenal cells in vitro. PATIENT AND RESULTS: A boy diagnosed with AAAS presented with short stature and increased oxidative stress in vivo assessed by increased thiobarbituric acid reactive substances (TBARS), which are markers of lipid peroxidation, and by the susceptibility of LDL to oxidation and the capacity of HDL to prevent it. A homozygous missense germline mutation (c.523G>T, p.Val175Phe) in AAAS was identified. N-acetylcysteine (600 mg orally, twice daily) decreased oxidative stress but did not change the patient's growth pattern. CONCLUSIONS: An increase in oxidative stress is reported for the first time in vivo in an AAAS patient. N-acetylcysteine was capable of decreasing TBARS levels, reducing the susceptibility of LDL to oxidation and improving the antioxidant role of HDL. The long-term effect of antioxidant treatment should be evaluated to determine the real benefit for the prevention of the degenerative process in AAAS.

What Is the Optimal Target Convective Volume in On-Line Hemodiafiltration Therapy?

Conventional diffusion-based dialysis modalities including high-flux hemodialysis are limited in their capacity to effectively remove large uremic toxins and to improve outcomes for end-stage chronic kidney disease (ESKD) patients. By increasing convective solute transport, hemodiafiltration (HDF) enhances solute removal capacity over a broad range of middle- and large-size uremic toxins implicated in the pathophysiology of chronic kidney disease. Furthermore, by offering flexible convection volume, on-line HDF permits customizing the treatment dose to the patient's needs. In addition, convective-based modalities have been shown to improve hemodynamic stability and to reduce patients' inflammation profile - both of which are implicated in CKD morbidity and mortality. Growing clinical evidence indicates that HDF-based modalities provide ESKD patients with a number of clinical and biological benefits, including improved outcomes. Interestingly, it has recently emerged that the clinical benefits associated with HDF are positively associated with the total ultrafiltered volume per session (and per week), namely convective dose. In this chapter, we revisit the concept of convective dose and discuss the threshold value above which an improvement in ESKD patient outcome can be expected. This particular point will be addressed by stratifying the level of efficacy of convective volumes, schematically defined as minimal, optimal, personalized, and maximal. In addition, factors and best clinical practices implicated in the achievement of an optimal convective dose are reviewed. To conclude, we show how HDF differs from standard hemodialysis and why HDF offers a paradigm shift in renal replacement therapy.

A novel TRAPPC11 mutation in two Turkish families associated with cerebral atrophy, global retardation, scoliosis, achalasia and alacrima.

BACKGROUND: Triple A syndrome (MIM #231550) is associated with mutations in the AAAS gene. However, about 30% of patients with triple A syndrome symptoms but an unresolved diagnosis do not harbour mutations in AAAS. OBJECTIVE: Search for novel genetic defects in families with a triple A-like phenotype in whom AAAS mutations are not detected. METHODS: Genome-wide linkage analysis, whole-exome sequencing and functional analyses were used to discover and verify a novel genetic defect in two families with achalasia, alacrima, myopathy and further symptoms. Effect and pathogenicity of the mutation were verified by cell biological studies. RESULTS: We identified a homozygous splice mutation in TRAPPC11 (c.1893+3A>G, [NM_021942.5], g.4:184,607,904A>G [hg19]) in four patients from two unrelated families leading to incomplete exon skipping and reduction in full-length mRNA levels. TRAPPC11 encodes for trafficking protein particle complex subunit 11 (TRAPPC11), a protein of the transport protein particle (TRAPP) complex. Western blot analysis revealed a dramatic decrease in full-length TRAPPC11 protein levels and hypoglycosylation of LAMP1. Trafficking experiments in patient fibroblasts revealed a delayed arrival of marker proteins in the Golgi and a delay in their release from the Golgi to the plasma membrane. Mutations in TRAPPC11 have previously been described to cause limb-girdle muscular dystrophy type 2S (MIM #615356). Indeed, muscle histology of our patients also revealed mild dystrophic changes. Immunohistochemically, ß-sarcoglycan was absent from focal patches. CONCLUSIONS: The identified novel TRAPPC11 mutation represents an expansion of the myopathy phenotype described before and is characterised particularly by achalasia, alacrima, neurological and muscular phenotypes.