Krabbe disease: Clinical, biochemical and molecular information on six new patients and successful retrospective diagnosis using stored newborn screening cards
Highlights
► Clinical, biochemical, molecular information on six patients with Krabbe disease. ► One patient presented with hydrocephalus. ► Retrospective galactocerebrosidase analysis in NBS samples stored up to 13 years. ► NBS test demonstrated 100% sensitivity and specificity. ► Five previously unreported mutations and 2 novel variants identified in GALC gene.
Introduction
Krabbe disease (OMIM #245200) or globoid-cell leukodystrophy is a severe neurodegenerative disorder caused by deficiency of galactocerebrosidase (GALC), a lysosomal enzyme responsible for the degradation of certain galactolipids found in myelin [1], [2], [3], [4]. Early infantile Krabbe disease presents with sudden onset irritability, startle response to stimuli and developmental arrest between three and six months of age in a previously healthy infant [5], [6]. Over the course of several months, affected infants develop hypertonicity, seizures and blindness and most do not survive past their second birthday [7]. Feeding difficulties and aspiration pneumonia are frequent complications. Krabbe disease may present later in infancy, in childhood, or even adulthood. Children with late infantile Krabbe disease present after 6 months of age with symptoms similar to the early infantile form, followed by rapid disease progression and death within two years [8]. Juvenile Krabbe disease usually presents acutely with ataxia or spastic paresis between three and eight years of age, followed by a slowly progressive disease course [8], [9], [10], [11], [12], [13]. Intellect, often intact at the onset of symptoms, may deteriorate or remain stable for years to decades after symptoms begin. A high incidence of late-onset Krabbe disease has been reported in Southern Italy [13].
The diagnosis of Krabbe disease may be aided by neuroimaging, nerve conduction studies and/or spinal tap. Cerebral spinal fluid protein is increased [5]. Diffuse, symmetric cerebral atrophy and demyelination is evident by brain CT and/or MRI [7]. Hydrocephalus has been reported in only three cases [14], [15], [16]. Peripheral neuropathy, demonstrated by reduced nerve conduction velocities, is evident in all cases of early infantile Krabbe disease and some patients with later onset [17].
In symptomatic individuals, the diagnosis of Krabbe disease can be made by enzyme analysis of GALC activity in leukocytes or cultured skin fibroblasts. Affected patients usually have severely decreased activity levels ranging from 0 to 5% of the normal mean; there is no correlation, however, between residual enzyme activity and disease severity [4], [18]. Leukocyte GALC enzyme activities of 8–20% in individuals without any of the classical presentations of Krabbe are inconclusive and require molecular confirmation.
The cDNA encoding GALC was cloned by Chen et al. in 1993, permitting molecular analysis and investigation of possible genotype–phenotype correlations [19]. Among early infantile patients, a common 30-kb deletion beginning in intron 10 and continuing beyond the end of the gene (c.502T/del) occurs with an allele frequency of 40–45% in patients of Northern European descent and 35% in patients from Mexico and South America with Spanish ancestry [4], [18]. The frequency of this deletion is highest (75% of mutant alleles) among patients from Sweden, where it likely originated [7]. Homozygosity for the 30-kb deletion or compound heterozygosity for this deletion and another severe mutation is always associated with early infantile Krabbe disease [18]. Two point mutations, c.1538C > T (p.T513M) and c.1652A > C (p.Y551S), make up another 10–15% of mutant alleles among infantile patients of Northern European descent [7], [18]. A substitution of aspartic acid for glycine at position 270 (c.809G > A) has only been found in cases of late-onset, although clinically variable, disease, regardless of the second mutation [4].
Treatment for Krabbe disease is currently limited to pre-symptomatic hematopoetic stem cell transplantation (HSCT) [20], [21]. With the promise of pre-symptomatic HSCT and the advent of assays to detect lysosomal enzyme deficiencies in newborn dried blood spots, newborn screening programs began to consider the addition of Krabbe disease [22], [23]. In 2006, New York became the first state to screen for the disorder, using measurement of GALC enzyme activity in dried newborn blood spots, followed by enzyme activity in leukocytes/molecular analysis, when indicated. Although diagnosis of the disease in the newborn period can be achieved, significant challenges remain to select patients who will benefit from HSCT and to predict post-transplant neurodevelopmental outcome [24], [25], [26], [27], [28]. While further investigation into the long term outcome of pre-symptomatic HSCT for Krabbe disease is needed, other benefits of newborn screening for the disorder are apparent. These include avoidance of diagnostic delay and the recognition of couples at risk who may benefit from available reproductive options. Recently, the state of Illinois also began screening for Krabbe disease. It is likely that other programs will do so in the future.
The sensitivity of GALC enzyme analysis in newborn screening samples obtained from known affected individuals, however, has not been validated. If this is a reliable screening test for Krabbe disease, one would expect to identify many pre-symptomatic infants and, therefore, correlations between genotype and clinical outcome are needed.
The purpose of this report is to present clinical, biochemical and genetic information on six patients with Krabbe disease not previously described in the literature, as well as to assess the sensitivity of GALC enzyme analysis in newborn dried blood spots obtained from affected individuals.
Section snippets
Patients
Between 2003 and 2008, five patients were diagnosed with Krabbe disease by the Metabolic Division at CHOC Children's (Orange, California). An additional patient with late onset disease and prolonged survival was referred to the Division during this time.
Enzyme analysis
Analysis of GALC activity in leukocytes was performed on all patients and on the parents of patients 1, 2 and 5 at the laboratory of David A. Wenger, PhD, (Lysosomal Diseases Testing Laboratory, Jefferson Medical College, Philadelphia, PA) using
Patient 1
Patient 1, the second child born to his parents, was delivered at 37 weeks gestation by emergency cesarean section due to decreased fetal movements. A nuchal cord was noted at birth; however, the patient had good Apgar scores and no perinatal complications. From birth, he was noted to have difficulty with breast feeding and taking formula. Weight and length were below the 5th centile, but followed a parallel curve. Early developmental milestones were normal. At 14 months, a clumsy, unsteady gait
Discussion
In this paper, we present six patients with Krabbe disease: three with early infantile disease, one of whom had hydrocephalus, two with late infantile onset and one who became symptomatic in childhood and had a protracted disease course. Patients 1 and 2 presented in late infancy (> 6 months of age), with subsequent rapid progression of neurological symptoms and death occurring two to three years after diagnosis. Patient 3 presented after two years of seemingly normal development with acute-onset
Acknowledgments
The authors wish to thank the Commission for Families and Children of Orange County for their support with the clinical work.
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