Atypical case of Wolfram syndrome revealed through targeted exome sequencing in a patient with suspected mitochondrial disease

In January 2008, a 61-year-old man with a history notable for diabetes mellitus (DM), autonomic neuropathy, diffuse brain atrophy, optic nerve atrophy (OA), and profound amnesia was referred to us to establish neurologic care. The patient carried a diagnosis of multiple system atrophy- cerebellar type (MSAc), principally because of severe cerebellar and brainstem atrophy on MRI.

The patient's early history was remarkable only for childhood bedwetting and urinary urgency as a young adult. He was otherwise well during this time and was a talented athlete who completed college and practiced as an accountant. In his early 20s, he developed bladder dysfunction of unclear etiology requiring intermittent straight catheterization, as well as erectile dysfunction.

At age 33, he was diagnosed with DM, presumed to be type 1, and began treatment with insulin therapy. Although there is no biochemical data available from the time of his original diagnosis, recent testing demonstrated a random C-peptide level of 0.6 ng/mL (reference range 0.9 to 4.3 ng/mL) at a time when his blood glucose was 83 mg/dL. He takes an average of 24 units of insulin per day, and has had good glycemic control with hemoglobin A1c measurements ranging between 6.5 and 7.2% over the last several years. He has had no evidence of retinopathy, or other microvascular or macrovascular complications. He had polyuria and polydipsia at the time of his initial DM diagnosis, but these symptoms resolved once he initiated insulin therapy.

The patient began dressing in strange colors in his 30s, and color blindness was ultimately diagnosed in his 40s. At age 53, the patient presented for a routine screening ophthalmology exam and was discovered to have bilateral OA with preserved vision. Brain MRI at that time revealed severe atrophy of the cerebellar hemispheres and vermis, pons, and middle cerebellar peduncles as well as moderate cerebral atrophy; a more recent study at age 61 showed these findings as well as more severe cerebral atrophy (Figure 1). Despite these radiographic findings, the patient and his wife reported no gait instability or upper extremity incoordination.

During his late 50s, the patient's neurologic status deteriorated. Formal neuropsychological evaluation revealed profound anterograde amnesia, with additional impairments in cognitive flexibility, executive function, naming, and high order visual processing skills. Attention span, mental tracking, verbal abstract reasoning, complex auditory instructions, and visual spatial functions were preserved. From a psychiatric perspective, he developed symptoms of depression, which responded to treatment with sertraline.

In parallel with the decline in his memory, the patient also developed progressive autonomic neuropathy, with gastroparesis and severe postural hypotension. The autonomic dysfunction exceeded what might be expected from his diabetes mellitus, given his good glycemic control and the absence of other diabetic complications. His bladder dysfunction worsened and he required suprapubic catheter placement at the age of 61. Due to his multiple functional deficits, the patient became unable to work and is now completely reliant upon his wife for care.

Regarding his family history, the patient was born to Ashkenazi Jewish parents and there was no parental consanguinity. His mother died from melanoma, and his father died from multiple strokes and a myocardial infarction. He has two adult daughters, one of whom has attention deficit hyperactivity disorder (ADHD) and Tourette syndrome, while the other suffers from chronic urinary tract infections. His maternal grandmother had type 2 DM, and a maternal first cousin had type 1 DM. No other close relatives have suffered from endocrine, psychiatric, or neurologic disease.

On physical examination, he appeared generally medically well. He weighed 79 kilograms and was 178 cm tall, yielding a body mass index of 25. Postural hypotension was evident with systolic blood pressure falling from 150 to 95 after one minute of standing, though asymptomatic. Funduscopic examination revealed optic atrophy bilaterally with no sign of diabetic retinopathy. Visual acuity was 20/40 in each eye. Pupil responses to light and accommodation were normal. Eye movements were normal with the exception of saccadic intrusion into horizontal smooth pursuit. Clinical examination revealed high tone hearing loss bilaterally. Audiometry demonstrated moderate sensorineural hearing loss in the high frequencies on the left, and mild sensorineural hearing loss in the mid-frequencies on the right sloping to a severe loss in the high frequencies (Figure 2). Word recognition was excellent in both ears; 98% on the right and 96% on the left. Muscle tone in the extremities was normal, bulk was intact, and strength was full. There was no evidence of dysmetria with finger-to-nose and heel-to-shin testing, and gait was slow but stable. His affect was flat and he was passive throughout the interview, speaking only when spoken to. He was not oriented to time or place. He could repeat four words, but could not learn them despite multiple attempts. He was unable to provide information concerning major current political or national news. He could, however, recall sizable fragments of remote memory from his college years.

The absence of the cerebellar motor syndrome and the presence of a profound amnestic syndrome on examination called the patient's diagnosis of MSAc into question [6], and we undertook re-evaluation of his case to explore alternate diagnoses. His laboratory work-up revealed an undetectable thiamine level, a surprising finding given his normal diet and the absence of alcohol abuse. We ascribed his amnestic disorder to presumed long-standing thiamine deficiency, but repletion produced minimal clinical impact. The involvement of multiple systems suggested the possibility of a mitochondrial disorder. Genetic testing for OPA1, MELAS, MERFF, LHON and NARP were negative, however analysis of mitochondrial DNA (mtDNA) from a muscle biopsy sample by both Southern blotting and PCR analysis revealed multiple heteroplasmic deletions. Biochemical testing revealed a minor defect in complex I of the electron transport chain. COX and SDH staining of the muscle biopsy specimen were unremarkable, and the mitochondria appeared grossly normal on electron microscopic examination. Occasional central vacuoles and tubular aggregates were seen in the myocytes, which were felt to be consistent with a mild non-specific myopathy.

Given the diagnostic uncertainty and concern for a mitochondrial disorder, the patient was enrolled in the mitochondrial disease registry at Massachusetts General Hospital. As part of this program, a sample of the patient's DNA from whole blood underwent targeted exome ("MitoExome") sequencing. Mitochondrial DNA and the exons of 1,600 nuclear genes either encoding mitochondrial proteins or implicated in Mendelian disorders with multi-system phenotypes were targeted using hybrid selection [7]. Amplified targets were sequenced on the Illumina GAIIx platform. Rare, protein-modifying variants found to be homozygous or potentially compound heterozygous were prioritized (Figure 3), revealing an X-linked functional polymorphism c.937G > T (p.D313Y) in GLA that is not considered pathogenic [8] and a homozygous c.1672C > T (p.R558C) missense mutation in exon 8 of WFS1 that has previously been reported in a patient with Wolfram syndrome [9]. No heteroplasmic mtDNA deletions were detected in whole blood. The patient's WFS1 mutation was verified through Sanger sequencing in a CLIA-certified laboratory, though not without complications; the initial report came back negative and only after requested follow-up was the homozygous mutation detected, thereby confirming the diagnosis of Wolfram syndrome.