Manuela Krumbholz

Three siblings with triple A syndrome with a novel frameshift mutation in the AAAS gene and a review of 17 independent patients with the frequent p.Ser263Pro mutation

The triple A syndrome is an autosomal recessive disorder characterized by adrenal insufficiency, alacrima, achalasia, and impairment of the central, peripheral, and autonomic nervous system functions. The disease is caused by mutations in the AAAS gene on chromosome 12q13 encoding the nuclear pore protein ALADIN. In the present study, we report three siblings with triple A syndrome caused by a compound heterozygous mutation consisting of a novel Val421 frameshift mutation in exon 14 and a previously described Ser236Pro (T>C transition) missense mutation in exon 8. The second mutation is one of the most frequent mutations in the AAAS gene, occurring in 17 independent patients from different countries. With haplotype analysis, we demonstrate a founder effect for at least 13 of the 17 patients. We conclude that, although very helpful in establishing the final diagnosis of triple A syndrome, DNA analysis is not useful for the prediction of the clinical expression and outcome of the disorder. Further investigations are necessary to evaluate the correlation between genotype and clinical phenotype in the triple A syndrome.

Intracellular ROS level is increased in fibroblasts of triple A syndrome patients

Triple A syndrome is named after the main symptoms of alacrima, achalasia, and adrenal insufficiency but also presents with a variety of neurological impairments. To investigate the causes of progressive neurodegeneration, we examined the oxidative status of fibroblast cultures derived from triple A syndrome patients in comparison to control cells. Patient cells showed a 2.1-fold increased basal level of reactive oxygen species (ROS) and a massive boost after induction of artificial oxidative stress by paraquat. We examined the expression of the ROS-detoxifying enzymes superoxide dismutase 1 and 2 (SOD1, SOD2), catalase, and glutathione reductase. The basal expression of SOD1 was significantly (1.3-fold) increased, and the expression of catalase was 0.7-fold decreased in patient cells after induction of artificial oxidative stress. We show that the mitochondrial network is 1.8-fold more extensive in patient cells compared to control fibroblasts although the maximal ATP synthesis was unchanged. Despite having the same energy potential as the controls, the patient cells showed a 1.4-fold increase in doubling time. We conclude that fibroblasts of triple A patients have a higher basal ROS level and an increased response to artificially induced oxidative stress and undergo “stress-induced premature senescence”. The increased sensitivity to oxidative stress may be a major mechanism for the neurodegeneration in triple A syndrome.

Cluster analysis of genomic ETV6–RUNX1 (TEL–AML1) fusion sites in childhood acute lymphoblastic leukemia

Fusion between ETV6 and RUNX1 defines the largest genetic subgroup in childhood ALL. The genomic fusion site, unique to individual patients and specific for the malignant clone, represents an ideal molecular marker for quantification of minimal residual disease. Sequencing of DNA breakpoints has been difficult due to the extended size of the respective breakpoint cluster regions. We therefore evaluated a specially designed multiplex long-range PCR assay in 65 diagnostic bone marrow samples for its suitability in routine use. Resulting fusion sites and breakpoints derived from previous studies were subject to cluster analysis to identify potential sequence motifs involved in translocation initiation.

Genomic BCR‐ABL1 breakpoints in pediatric chronic myeloid leukemia

Chronic myeloid leukemia (CML) is a rare disease in children and adolescents and various aspects—from molecular genesis to therapy regimen—have been taken over from studies on the more prevalent adult CML. However, differences have been observed between malignancies with identical underlying chromosomal translocations, but occurring at different age groups, suggesting some diversity in the mechanisms of formation and leukemogenesis. A multiplex long‐range PCR‐based assay was developed to allow fast and reliable amplification of patient‐specific BCR‐ABL1 fusion sequences from genomic DNA. The localization of breakpoints was analyzed with respect to distribution within the breakpoint cluster regions, sequence features, and association to repetitive elements or motifs associated with DNA recombination. The genomic fusion sites of 59 pediatric CML patients showed a bimodal breakpoint distribution in BCR that was different from the distribution in adult CML cases, but with similarities to BCR‐ABL1‐positive, acute lymphoblastic leukemia in adults. BCR breakpoints were found more frequently positioned within, or close to, Alu repeats than would be expected based on their overall sequence proportion. Technical aspects of the highly sensitive DNA‐based quantification of residual CML cells by specific fusion sequence during tyrosine kinase inhibitor therapy are exemplified in a subcohort of pediatric CML patients.

Axonal neuropathy with unusual pattern of amyotrophy and alacrima associated with a novel AAAS mutation p.Leu430Phe

The triple A syndrome is caused by autosomal recessively inherited mutations in the AAAS gene and is characterized by achalasia, alacrima and adrenal insufficiency as well as progressive neurological impairment. We report on a 14-year-old girl with slowly progressive axonal motor neuropathy with conspicuous muscle wasting of hypothenars and calves as well as alacrima. The mutation analysis of the AAAS gene revealed a compound heterozygous mutation: a c.251G>A mutation in exon 2 that had been reported previously, and a novel c.1288C>T mutation in exon 14. At the transcriptional level, the c.251G>A transition results in an aberrant splicing and decay of this RNA strand so that the particular clinical picture results from the novel c.1288C>T, (p.Leu430Phe, L430F) mutation in a hemizygous form. With transfection experiments, we demonstrate that GFP–ALADINL430F correctly localizes to nuclear pore complexes. Therefore, we conclude that this point mutation impairs ALADIN function at the nuclear pore.

Late-Onset Triple A Syndrome: A Risk of Overlooked or Delayed Diagnosis and Management

Background/Aims: A 33-year-old man was referred for the first time to the Division of Neurology because of the presence and progression of neurological symptoms. Dysphagia, weakness, reduced tear production, and nasal speech were present. In order to point the attention of late-onset triple A syndrome we describe this case and review the literature. Methods: Hormonal and biochemical evaluation, Schirmer test, tilt test and genetic testing for AAAS gene mutations. Results: Late-onset triple A syndrome caused by a novel homozygous missense mutation in the AAAS gene (A167V in exon 6) was diagnosed at least 17 years after symptom onset. Conclusions: The association between typical signs and symptoms of triple A syndrome should suggest the diagnosis even if they manifest in adulthood. The diagnosis should be confirmed by Schirmer test, endocrine testing (both basal and dynamic), genetic analysis, and detailed gastroenterological and neurological evaluations. Awareness of the possible late onset of the disease and of diagnosis in adulthood is still poor among clinicians, the acquaintance with the disease is more common among pediatricians. The importance of an adequate multidisciplinary clinical approach, dynamic testing for early diagnosis of adrenal insufficiency and periodical reassessment of adrenal function are emphasized.

Triple A syndrome mimicking ALS

We report a 22-year-old female who presented with distal muscular atrophy and weakness in all limbs for two years. Reflexes were symmetrically brisk and electrodiagnostic studies were consistent with upper and lower motor neuron involvement. A diagnosis of juvenile ALS was considered. However, surgery for achalasia in childhood and identification of alacrima and adrenal insufficiency suggested Triple A syndrome accompanied by neurological symptoms. Sequencing of the AAAS gene identified compound heterozygous mutations confirming the clinical diagnosis and demonstrating that Triple A syndrome can mimic juvenile ALS.

Genomic EWS-FLI1 fusion sequences in Ewing sarcoma resemble breakpoint characteristics of immature lymphoid malignancies.

Chromosomal translocations between the EWS gene and members of the ETS gene family are characteristic molecular features of the Ewing sarcoma. The most common translocation t(11;22)(q24;q12) fuses the EWS gene to FLI1, and is present in 85–90% of Ewing sarcomas. In the present study, a specifically designed multiplex long-range PCR assay was applied to amplify genomic EWS-FLI1 fusion sites from as little as 100 ng template DNA. Characterization of the EWS-FLI1 fusion sites of 42 pediatric and young adult Ewing sarcoma patients and seven cell lines revealed a clustering in the 5′ region of the EWS-breakpoint cluster region (BCR), in contrast to random distribution of breakpoints in the FLI1-BCR. No association of breakpoints with various recombination-inducing sequence motifs was identified. The occurrence of small deletions and duplications at the genomic junction is characteristic of involvement of the non-homologous end-joining (NHEJ) repair system, similar to findings at chromosomal breakpoints in pediatric leukemia and lymphoma.

Bimodal distribution of genomic MLL breakpoints in infant acute lymphoblastic leukemia treatment

Bimodal distribution of genomic MLL breakpoints in infant acute lymphoblastic leukemia treatment

Sustained complete molecular remission after imatinib discontinuation in children with chronic myeloid leukemia

Approximately 40% of adults with chronic myeloid leukemia (CML) in prolonged complete molecular response (CMR) remain in CMR after imatinib discontinuation. Corresponding information in children is lacking. Two children with CML in CMR for 48 and 19 months after imatinib discontinuation showed low‐level fluctuating disease at RNA transcript and genomic DNA levels. Both patients were low risk according to adult criteria. Since adults with molecular relapse responded to re‐introduction of imatinib, we postulated that treatment discontinuation in low risk children might be justified within clinical trials with close monitoring. This may help to minimize exposure to imatinib and its potential side effects. Pediatr Blood Cancer 2014;61:2080–2082.