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.

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.

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.

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.

"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.

Mutations in GMPPA cause a glycosylation disorder characterized by intellectual disability and autonomic dysfunction.

In guanosine diphosphate (GDP)-mannose pyrophosphorylase A (GMPPA), we identified a homozygous nonsense mutation that segregated with achalasia and alacrima, delayed developmental milestones, and gait abnormalities in a consanguineous Pakistani pedigree. Mutations in GMPPA were subsequently found in ten additional individuals from eight independent families affected by the combination of achalasia, alacrima, and neurological deficits. This autosomal-recessive disorder shows many similarities with triple A syndrome, which is characterized by achalasia, alacrima, and variable neurological deficits in combination with adrenal insufficiency. GMPPA is a largely uncharacterized homolog of GMPPB. GMPPB catalyzes the formation of GDP-mannose, which is an essential precursor of glycan moieties of glycoproteins and glycolipids and is associated with congenital and limb-girdle muscular dystrophies with hypoglycosylation of α-dystroglycan. Surprisingly, GDP-mannose pyrophosphorylase activity was unchanged and GDP-mannose levels were strongly increased in lymphoblasts of individuals with GMPPA mutations. This suggests that GMPPA might serve as a GMPPB regulatory subunit mediating feedback inhibition of GMPPB instead of displaying catalytic enzyme activity itself. Thus, a triple-A-like syndrome can be added to the growing list of congenital disorders of glycosylation, in which dysregulation rather than mere enzyme deficiency is the basal pathophysiological mechanism.

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.

A founder mutation in Anoctamin 5 is a major cause of limb-girdle muscular dystrophy.

The limb-girdle muscular dystrophies are a group of disorders with wide genetic and clinical heterogeneity. Recently, mutations in the ANO5 gene, which encodes a putative calcium-activated chloride channel belonging to the Anoctamin family of proteins, were identified in five families with one of two previously identified disorders, limb-girdle muscular dystrophy 2L and non-dysferlin Miyoshi muscular dystrophy. We screened a candidate group of 64 patients from 59 British and German kindreds and found the truncating mutation, c.191dupA in exon 5 of ANO5 in 20 patients, homozygously in 15 and in compound heterozygosity with other ANO5 variants in the rest. An intragenic single nucleotide polymorphism and an extragenic microsatellite marker are in linkage disequilibrium with the mutation, suggesting a founder effect in the Northern European population. We have further defined the clinical phenotype of ANO5-associated muscular dystrophy. Patients show adult onset proximal lower limb weakness with highly raised serum creatine kinase values (average 4500 IU/l) and frequent muscle atrophy and asymmetry of muscle involvement. Onset varies from the early 20 s to 50 s and the weakness is generally slowly progressive, with most patients remaining ambulant for several decades. Distal presentation is much less common but a milder degree of distal lower limb weakness is often observed. Upper limb strength is only mildly affected and cardiac and respiratory function is normal. Females appear less frequently affected. In the North of England population we have identified eight patients with ANO5 mutations, suggesting a minimum prevalence of 0.27/100,000, twice as common as dysferlinopathy. We suggest that mutations in ANO5 represent a relatively common cause of adult onset muscular dystrophy with high serum creatine kinase and that mutation screening, particularly of the common mutation c.191dupA, should be an early step in the diagnostic algorithm of adult limb-girdle muscular dystrophy patients.

Long-term clinical follow-up and molecular genetic findings in eight patients with triple A syndrome.

UNLABELLED: The triple A syndrome (Allgrove syndrome, OMIM #231550) is caused by autosomal recessively inherited mutations in the AAAS gene on chromosome 12q13 encoding the nuclear pore protein ALADIN. This multisystemic disease is characterised by achalasia, alacrima, adrenal insufficiency and neurological impairment. We analyse long-term clinical follow-up and results of sequencing of the AAAS gene in eight patients with triple A syndrome aged from 2 to 35 years. At the time of diagnosis, all patients presented with alacrima, neurological dysfunction, dermatological abnormalities, seven of them with adrenal insufficiency and five of them with achalasia. Sequencing of the AAAS gene identified the p.S263P mutation in five of eight patients, supporting the hypothesis that this mutation is a founder mutation in Slavic population. One of the patients is homozygous for the p.S263P mutation, two are compound heterozygous for the p.S263P and the p.G14fs mutation, two are compound heterozygous for the p.S263Pro mutation and p.S296Y mutation, two are compound heterozygous for the p.G14fs and the p.Q387X mutations and one is homozygous for the p.Q387X mutation. In the course of the follow-up time of 4-29 years, progression of existing and appearance of new symptoms developed. Although severe, many of these symptoms presented in all six young adult patients are often overlooked or neglected: postural hypotension with blurred vision and syncope, hyposalivation resulting with complete edentulosis, talocrular contractures with permanent walking difficulties and erectile dysfunction in male patients. Triple A syndrome is a progressive debilitating disorder which may seriously affect quality of life and even be life-threatening in patients with severe neurological impairment. CONCLUSION: Long-term follow-up of patients with triple A syndrome revealed a variety of the clinical features involving many systems. Progressive natural course of the disease may seriously affect quality of life and even be life-threatening in patients with severe neurological impairment.

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.

Two siblings with triple A syndrome and novel mutation presenting as hereditary polyneuropathy.

The clinical and molecular data on triple A syndrome in two siblings (girl 3.5 years and boy 5.5 years at presentation) with early onset of neurological dysfunction are described. Both patients showed delayed developmental milestones and neurological dysfunctions (motor and sensory demyelinating neuropathy, marked hyperreflexia, calves hypothrophy, pes cavus, gait disturbance) in early childhood, when erroneously diagnosed with hereditary polyneuropathy, most likely Charcot-Marie-Tooth disease. After a severe adrenal crisis in the younger sister at the age of 3 years, the older brother aged 5.5 years was also evaluated and latent adrenal insufficiency was discovered. As both of the siblings had alacrima, hyperkeratosis of palms, cutis anserina, and nasal speech, diagnosis of triple A syndrome was considered. Sequencing of the AAAS gene detected a compound heterozygous mutation consisting of a novel mutation p.Ser296Tyr (c.887C>A) in exon 9 and a previously described p.Ser263Pro (c.787T>C) missense mutation in exon 8 in both siblings. In conclusion, triple A syndrome should be considered in patients presenting with early neurological dysfunction and developmental delay. Alacrima as the earliest and most consistent clinical sign should be investigated by Schirmer test. Patients should be regularly tested for adrenal dysfunction to prevent life-threatening adrenal crises.

New ophthalmic features in a family with triple A syndrome.

We report three subjects of a Greek family affected by triple A syndrome (AAAS). All patients underwent complete ophthalmic examination, full-field electroretinogram (ERG), visual evoked responses (VER), optical coherence tomography (OCT) and molecular analysis of the AAA gene. All patients had alacrima. In two of them, the proband and her brother, bilateral optic atrophy was assessed and the VER were pathological. In contrast, the ERG was normal. OCT showed a decrease of the retinal nerve fiber layer. The third case had only alacrima and the optic nerves were normal. The molecular genetic study of the AAAS gene revealed a homozygous missense mutation p.Ala167Val. To our knowledge this is the first time a family with AAAS has been investigated using OCT, VER and ERG. Our findings illustrate that the retina is not involved. There is also an interfamilial variability concerning the involvement of the optic nerves.

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.

Triple A syndrome: 32 years experience of a single centre (1977-2008).

Triple A syndrome is an autosomal recessive disorder characterized by alacrima, achalasia, ACTH-resistant adrenal insufficiency, autonomic dysfunction, and neurodegeneration. Mutations in the AAAS gene on chromosome 12q13 encoding the nuclear pore protein ALADIN have been reported in these patients. Over the period 1977-2008 we evaluated ten subjects with the clinical diagnosis of triple A syndrome. Molecular analysis was performed in seven patients and revealed that all except one are compound heterozygotes for two mutations in the AAAS gene. Two novel mutations were detected: c.123+2T>C resulted in splice defect while c.1261_1262insG mutation resulted in a truncated protein (p.V421fs), which most probably is not functional. Genotype-phenotype correlation could not be established. In all our patients, except one sibling of previously diagnosed brother and sister, genetic analysis was performed when at least two symptoms were present, usually alacrima and achalasia. Based on our experience, we recommend that in case of the presence of alacrima and at least one more symptom of triple A syndrome, adrenal function testing and molecular analysis should be performed. In all children with mutation in AAAS gene, regular follow up of adrenal function is necessary to avoid adrenal crisis and start substitution therapy as soon as adrenal insufficiency is noted.

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.

The nuclear pore complex protein ALADIN is anchored via NDC1 but not via POM121 and GP210 in the nuclear envelope.

The nuclear pore complex (NPC) consists of approximately 30 different proteins and provides the only sites for macromolecular transport between cytoplasm and nucleus. ALADIN was discovered as a new member of the NPC. Mutations in ALADIN are known to cause triple A syndrome, a rare autosomal recessive disorder characterized by adrenal insufficiency, alacrima, and achalasia. The function and exact location of the nucleoporin ALADIN within the NPC multiprotein complex is still unclear. Using a siRNA-based approach we downregulated the three known membrane integrated nucleoporins NDC1, GP210, and POM121 in stably expressing GFP-ALADIN HeLa cells. We identified NDC1 but not GP210 and POM121 as the main anchor of ALADIN within the NPC. Solely the depletion of NDC1 caused mislocalization of ALADIN. Vice versa, the depletion of ALADIN led also to disappearance of NDC1 at the NPC. However, the downregulation of two further membrane-integral nucleoporins GP210 and POM121 had no effect on ALADIN localization. Furthermore, we could show a direct association of NDC1 and ALADIN in NPCs by fluorescence resonance energy transfer (FRET) measurements. Based on our findings we conclude that ALADIN is anchored in the nuclear envelope via NDC1 and that this interaction gets lost, if ALADIN is mutated. The loss of integration of ALADIN in the NPC is a main pathogenetic aspect for the development of the triple A syndrome and suggests that the interaction between ALADIN and NDC1 may be involved in the pathogenesis of the disease.

Mice lacking the nuclear pore complex protein ALADIN show female infertility but fail to develop a phenotype resembling human triple A syndrome.

Triple A syndrome is a human autosomal recessive disorder characterized by adrenal insufficiency, achalasia, alacrima, and neurological abnormalities affecting the central, peripheral, and autonomic nervous systems. In humans, this disease is caused by mutations in the AAAS gene, which encodes ALADIN, a protein that belongs to the family of WD-repeat proteins and localizes to nuclear pore complexes. To analyze the function of the gene in the context of the whole organism and in an attempt to obtain an animal model for human triple A syndrome, we generated mice lacking a functional Aaas gene. The Aaas-/- animals were found to be externally indistinguishable from their wild-type littermates, although their body weight was on the average lower than that of wild-type mice. Histological analysis of various tissues failed to reveal any differences between Aaas-/- and wild-type mice. Aaas-/- mice exhibit unexpectedly mild abnormal behavior and only minor neurological deficits. Our data show that the lack of ALADIN in mice does not lead to a triple A syndrome-like disease. Thus, in mice either the function of ALADIN differs from that in humans, its loss can be readily compensated for, or additional factors, such as environmental conditions or genetic modifiers, contribute to the disease.

Clinical and molecular genetic findings in a 6-year-old Bosnian boy with triple A syndrome.

The triple A syndrome is a rare autosomal recessive disease that is characterised by the triad of adrenocorticotropin (ACTH)-resistant adrenal insufficiency, achalasia and alacrima. In most patients, neurological and dermatological abnormalities are associated features. We report on the first Bosnian patient with triple A syndrome. Endocrine investigation confirmed primary adrenal insufficiency at the age of 5.8 years. Two months later, achalasia was diagnosed, and in the presence of alacrima, the patient satisfies the diagnostic criteria of triple A syndrome. In addition, a large number of associated neurological and dermatological features were present in this patient. Moreover, he has dysmorphic facial features, which have not been previously described in triple A syndrome. Triple A syndrome was confirmed by molecular analysis, revealing a nonsense mutation p.W84X in the AAAS gene. The parents are both heterozygous carriers of the mutation. The affected twin brother unfortunately died from hypoglycaemic shock, despite a normal cortisol rise in an ACTH stimulation test. Further, triple A syndrome patients carrying the identical homozygous p.W84X mutation have to be studied to assess a genotype-phenotype relationship for this mutation.