Mutations of the AAAS gene in an Indian family with Allgrove's syndrome

Abstract

The triple A or Allgrove's syndrome is an autosomal recessive disorder characterized by the triad of achalasia cardia, alacrima and ACTH resistant adrenocortical insufficiency. Mutations of the Achalasia-Addisonianism-Alacrima-Syndrome (AAAS) gene on chromosome 12q13 are associated with this syndrome. We report an Indian family where two siblings were homozygous for a known mutation of the AAAS gene and presented with the classical triad of symptoms. The mother and the brother were heterozygous and asymptomatic. The affected siblings had iron deficiency anemia and the younger sister had pes cavus and palmoplantar keratosis. Neurological symptoms were absent in both affected children. Recognition of this syndrome can lead to early treatment of adrenal insufficency and genetic counselling.

Key Words: Allgrove's syndrome; Triple A syndrome; Autosomal recessive; Genetic mutation; India

INTRODUCTION

Allgrove’s syndrome, also known as the triple A syndrome is characterized by the triad of adrenal insufficiency, achalasia cardia and alacrima[1]. Ever since its first description in 1978, more than 70 cases have been reported in the literature. The condition is associated with impairment of the central, peripheral and autonomic nervous systems, suggesting that it is a result of a mutation in a pleiotropic gene. Other reported abnormalities include palmoplantar hyperkeratosis, short stature, osteoporosis and microcephaly[2-4]. Progression of neurological symptoms has been reported with worsening of peripheral neuropathy, dementia, long tract degeneration, dysarthria and ataxia[5,6].

Pathological studies from esophageal myomectomy specimens of children afflicted with Allgrove’s syndrome show fibrosis of the intermuscular plane and absent myenteric ganglia[3]. Neuronal NO synthase has also been found to be deficient and could explain the poor relaxation of the lower esophageal sphincter (LES). Necropsy has been carried out in one of the pairs of original siblings published, which reveals atrophy of the adrenal cortical zona fasciculata and zona reticularis and absence of ganglion cells and nerve fibers in the lower esophagus[1].

This syndrome has been mapped to a novel gene on chromosome 12q 13 near the type II keratin gene cluster, and the gene is designated as the Achalasia-Addisonianism-Alacrima-Syndrome gene (the AAAS gene)[7]. There is a great deal of variability of symptoms and severity between patients with the same AAAS mutation, implying variable expression of this mutated gene.

This report describes the clinical presentation of two affected siblings with Allgrove’s syndrome and analyses the mutations in the immediate family.

CASE REPORT

A seventeen year old young lady presented along with her fifteen year old younger sister with identical symptoms of dysphagia, excessive fatigue and skin pigmentation. The elder sibling had been symptomatic for eight years and her sister for seven years respectively. Both had progressive difficulty in swallowing and regurgitation of food at the time of presentation. They had extreme tiredness and fatigue. The mother had noticed progressive pigmentation in both her daughters. The younger sibling had several episodes of sporadic abdominal pain and vomiting. Their milestones had been normal and they had attained menarche and thelarche normally. Their parents were non-consanguinous and their father had expired following an accident. One elder sister had died of meningitis at nine years of age and on questioning the mother, she mentioned the daughter having similar facial hyperpigmentation. A brother was asymptomatic at 16 years of age. Examination of both patients revealed normal blood pressure and heart rate with no postural changes. Both sisters had facial and palmar hyperpigmentation. The younger sibling had bilateral palmo-plantar keratosis and pes cavus. The conjunctiva and oral cavities of both sisters were dry. Schirmer’s test confirmed decreased lacrimation. Both were noted to have bilateral temporal pallor of optic discs.

Hemogram showed iron deficiency anemia in both sisters. Biochemical tests and chest X ray were normal. Barium swallow showed dilated esophagus and absent peristalsis in both sisters. Esophageal manometry done to confirm the diagnosis showed elevated LES pressures (45 mm and 60 mm respectively), poor relaxation to swallow and absent body peristalsis consistent with achalasia. Serum 8 am cortisol was 3.94 μg/deciliter and 3.37 μg/ deciliter in both sisters respectively and post Synacthen values were 5.16 μg/ deciliter and 4.10 μg/ deciliter respectively, consistent with Addison’s disease. The clinical features and laboratory investigations are summarized in Table 1.

Table 1 Summary of clinical features and laboratory investigation in both siblings with Allgrove’s syndrome.

Variable	Elder sibling	Younger sibling
Dysphagia	Present	Present
Generalized pigmentation	Present	Present
Abdominal pain	Absent	Present
Decreased lacrimation	Present	Present
Postural hypotension	Absent	Absent
Palmoplantar keratosis	Absent	Present
Temporal optical pallor	Present	Present
Hemoglobin (mg%)	6.8	8.1
Albumin (mg%)	4.2	4.2
Pre-Synacthen cortisol (μg/deciliter)	3.94	3.37
Post-Synacthen cortisol (μg/deciliter)	5.16	4.1
Barium swallow	Aperistaltic dilated esophagus	Aperistaltic dilated esophagus
LES pressure on manometry (mmHg)	45	60

The confirmation of achalasia, Addison’s disease, history of dry eye in both siblings, palmo-plantar hyperkeratosis in one sister and positive family history pointed to a clinical diagnosis of Allgrove’s syndrome. Molecular testing for the AAAS gene causing Allgrove’s syndrome was initiated to confirm the diagnosis. Blood samples from the patients, their mother and unaffected brother were taken after consent for AAAS gene analysis. Replacement therapy with corticosteroids and mineralocorticoids dramatically improved the symptoms in both the sisters. They have been advised to make frequent follow-up visits at the clinic.

MUTATION ANALYSIS

DNA was extracted using standard protocols. Coding sequences including exon-intron boundaries were amplified from genomic DNA and sequenced as reported previously [8]. Sequencing of the coding regions and exon/intron junctions of the AAAS gene in the two sisters revealed a homozygous splice mutation in exon 1, resulting in a frameshift after glycine at amino acid position 14 and a premature stopcodon (G14fs). This is the result of the transversion C to A at nucleotide position 43, forming a new premature splice donor site after G at nucleotide position 40 with a consecutive loss of the following 83 bp from exon 1. The mother is a heterozygote and so is the brother. Thus it verified the carrier status of the unaffected family members. This gene mutation analysis confirmed the diagnosis and helped in genetic counselling.

DISCUSSION

Triple A syndrome (Allgrove’s syndrome) is an autosomal recessive genetic disorder occurring due to mutations in the AAAS gene, resulting in phenotypic manifestations of achalasia, addisonianism, alacrima and variable symptoms of progressive neurological involvement. Using positional cloning, the product of the AAAS gene, designated ALADIN for alacrima, achalasia, adrenal insufficiency neurological disorder, was identified as a protein belonging to the WD repeat family[9]. Proteins belonging to this family have a wide functional diversity in that they are involved in signal transduction, RNA processing, vesicular trafficking, cytoskeleton assembly and cell division control[10].

The ubiquitous expression of AAAS is in accordance with the multisystemic character of the disorder and the predominant occurrence in the adrenal gland, gastrointestinal and nervous structures is in line with the tissues mainly affected in triple A syndrome. Various mutations have been described in patients from European, North African, Asian and American populations[8]. Ours is the first report of mutations in the AAAS gene in an Indian family with Allgrove’s syndrome. The transversion of C to A at nucleotide position 43 results in a markedly truncated protein, and it has been reported and confirmed that the mutation caused the disease[8].

Genotypic-phenotypic correlations have not been consistent in Allgrove’s syndrome[11]. We noted similarities in presentation in both these siblings. However, palmoplantar keratosis and pes cavus was present in only the younger sister despite both siblings having the same mutation. There are reports of patients presenting without hypoadrenalism, which is the hallmark of this syndrome[12]. From a large series of patients it has been seen that frameshift, stop codon and functionally significant mutations are likely to lead to a more severe phenotype, most probably occurring by a loss-of-function effect of the mutated protein[13]. Future studies need to focus on functional analysis of this mutated gene in order to comprehend the myriad presentations of the Allgrove’s syndrome.

ACKNOWLEDGMENTS

We acknowledge the cooperation of the patients and their immediate relatives for providing the samples.