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Molecular Genetics and Metabolism 105 (2012) 126–131

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Molecular Genetics and Metabolism

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Krabbe disease: Clinical, biochemical and molecular information on six new patients and successful retrospective diagnosis using stored cards

R.L. Puckett a, J.J. Orsini b, G.M. Pastores c, R.Y. Wang a, R. Chang a, C.A. Saavedra-Matiz b, P.A. Torres c, B. Zeng c, M. Caggana b, F. Lorey d, J.E. Abdenur a,⁎ a Division of Metabolic Disorders, CHOC Children's, Orange, CA, USA b Newborn Screening Program, New York State Department of Health, Albany, NY, USA c New York University School of Medicine, New York City, NY, USA d Genetic Disease Screening Program, California Department of Public Health, Richmond, CA, USA article info abstract

Article history: Purpose: To present clinical, biochemical and molecular information on six new clinically diagnosed Krabbe Received 21 August 2011 disease patients and assess the sensitivity of retrospective galactocerebrosidase measurement in their new- Received in revised form 18 October 2011 born screening samples. Accepted 18 October 2011 Methods: Medical records were reviewed. Galactocerebrosidase activity was measured in leukocytes and, ret- Available online 25 October 2011 rospectively, in the patients' newborn screening cards (stored for 1.4 to 13.5 years). GALC analysis was performed. Keywords: Results: Five patients with Krabbe disease, one of whom also had hydrocephalus, became symptomatic during Krabbe disease Newborn screening infancy. A sixth patient presented with and developmental regression at age two and had a pro- Galactocerebrosidase tracted disease course. Galactocerebrosidase activity in leukocytes ranged from 0.00 to 0.20 nmol/h/mg pro- Lysosomal storage diseases tein. Low galactocerebrosidase activity (range: 3.2% to 11.1% of the daily mean), consistent with Krabbe Bone marrow transplant disease, was detected in each of the newborn screening samples. GALC molecular analysis identified six pre- HSCT viously unreported and two novel sequence variants. Conclusion: Our cases highlight the clinical variability of Krabbe disease. Galactocerebrosidase activity in newborn dried blood spots is a highly sensitive test, even when samples have been stored for many years. The high frequency of private mutations in the GALC gene may limit the use of genetic information for making treatment decisions in the newborn period. © 2011 Elsevier Inc. All rights reserved.

1. Introduction form, followed by rapid disease progression and death within two years [8]. Juvenile Krabbe disease usually presents acutely with Krabbe disease (OMIM #245200) or globoid-cell or spastic paresis between three and eight years of age, followed by a is a severe neurodegenerative disorder caused by deficiency of slowly progressive disease course [8–13]. Intellect, often intact at the galactocerebrosidase (GALC), a lysosomal responsible for onset of symptoms, may deteriorate or remain stable for years to the degradation of certain galactolipids found in [1–4]. Early decades after symptoms begin. A high incidence of late-onset Krabbe infantile Krabbe disease presents with sudden onset , star- disease has been reported in Southern Italy [13]. tle response to stimuli and developmental arrest between three and The diagnosis of Krabbe disease may be aided by neuroimaging, six months of age in a previously healthy infant [5,6]. Over the course conduction studies and/or spinal tap. Cerebral spinal fluid pro- of several months, affected infants develop hypertonicity, seizures tein is increased [5]. Diffuse, symmetric and demye- and blindness and most do not survive past their second birthday lination is evident by CT and/or MRI [7]. Hydrocephalus has [7]. Feeding difficulties and aspiration pneumonia are frequent com- been reported in only three cases [14–16]. , plications. Krabbe disease may present later in infancy, in childhood, demonstrated by reduced nerve conduction velocities, is evident in or even adulthood. Children with late infantile Krabbe disease present all cases of early infantile Krabbe disease and some patients with after 6 months of age with symptoms similar to the early infantile 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 ⁎ Corresponding author at: CHOC Children's Division of Metabolic Disorders, 455 S Main St. Orange, CA 92868, USA. Fax: +1 714 532 8362. activity levels ranging from 0 to 5% of the normal mean; there is no E-mail address: [email protected] (J.E. Abdenur). correlation, however, between residual enzyme activity and disease

1096-7192/$ – see front matter © 2011 Elsevier Inc. All rights reserved. doi:10.1016/j.ymgme.2011.10.010 R.L. Puckett et al. / Molecular Genetics and Metabolism 105 (2012) 126–131 127 severity [4,18]. Leukocyte GALC enzyme activities of 8–20% in individ- substrate . Results were compared with that labora- uals without any of the classical presentations of Krabbe are inconclu- tory's reference ranges. sive and require molecular confirmation. The cDNA encoding GALC was cloned by Chen et al. in 1993, per- 2.3. Enzyme analysis of newborn dried blood spots mitting molecular analysis and investigation of possible genotype– phenotype correlations [19]. Among early infantile patients, a com- Dried blood spots were obtained on all patients for routine new- mon 30-kb deletion beginning in intron 10 and continuing beyond born screening. The remainder of each of the specimens was stored the end of the gene (c.502T/del) occurs with an allele frequency of at −20° with desiccant, and, after parental consent was obtained, 40–45% in patients of Northern European descent and 35% in patients shipped to the Krabbe Laboratory at the New York State Department from Mexico and South America with Spanish ancestry [4,18]. The of Health (Albany, New York) for retrospective measurement of GALC frequency of this deletion is highest (75% of mutant alleles) among activity. Thirty control samples (5 for each patient) obtained at the patients from Sweden, where it likely originated [7]. Homozygosity same time and stored in the same manner were also included. The for the 30-kb deletion or compound heterozygosity for this deletion laboratory was blind regarding the sample identity (patient or con- and another severe mutation is always associated with early infantile trol). A result is considered abnormal by the New York State Newborn Krabbe disease [18]. Two point mutations, c.1538C>T (p.T513M) and Screening Program when the GALC activity is b20% of the daily mean. c.1652A>C (p.Y551S), make up another 10–15% of mutant alleles Normally, the New York State protocol calls for re-testing of all abnor- among infantile patients of Northern European descent [7,18]. A sub- mal results in duplicate, followed by molecular analysis of samples stitution of aspartic acid for glycine at position 270 (c.809G>A) has with activity ≤12% of the daily mean. As this study was performed only been found in cases of late-onset, although clinically variable, retrospectively on a limited amount of sample from each patient, disease, regardless of the second mutation [4]. enzymatic analysis was performed only once, followed by molecular Treatment for Krabbe disease is currently limited to pre-symptomatic testing. hematopoetic stem cell transplantation (HSCT) [20,21]. With the promise of pre-symptomatic HSCT and the advent of assays to detect lysosomal 2.4. Mutation analysis enzyme deficiencies in newborn dried blood spots, newborn screening programs began to consider the addition of Krabbe disease [22,23].In Whole blood or isolated DNA from patients 2 to 6 and from the 2006, New York became the first state to screen for the disorder, using parents of patient 1 was sent to the Neurogenetics Laboratory at measurement of GALC enzyme activity in dried newborn blood spots, New York University (New York City, New York) for GALC mutation followed by enzyme activity in leukocytes/molecular analysis, when indi- analysis. Results were confirmed in the Krabbe Laboratory at the cated. Although diagnosis of the disease in the newborn period can be New York State Department of Health using DNA isolated from the achieved, significant challenges remain to select patients who will benefit newborn dried blood spots of all six patients. Polymerase chain reac- from HSCT and to predict post-transplant neurodevelopmental outcome tion and agarose gel electrophoresis are used by both laboratories to [24–28]. While further investigation into the long term outcome of pre- detect the common 30-kb deletion and, by New York State, to detect symptomatic HSCT for Krabbe disease is needed, other benefits of new- a recurrent 7.4-kb deletion. Prior to sequencing, the New York State born screening for the disorder are apparent. These include avoidance lab employed a LightCycler real-time polymerase chain reaction of diagnostic delay and the recognition of couples at risk who may benefit assay (Roche Applied Science, Indianapolis, IN), to test for three from available reproductive options. Recently, the state of Illinois also common GALC polymorphisms known to attenuate enzyme activity began screening for Krabbe disease. It is likely that other programs will (p.I546T, p.R168C, and p.D232N) and three common point mutations do so in the future. (p.T513M, p.Y551S, and c.1424delA). Both laboratories performed The sensitivity of GALC enzyme analysis in newborn screening semiautomated bidirectional DNA sequencing on all 17 exons and samples obtained from known affected individuals, however, has two promoter regions of the GALC gene. not been validated. If this is a reliable screening test for Krabbe dis- ease, one would expect to identify many pre-symptomatic infants 3. Results and, therefore, correlations between genotype and clinical outcome are needed. 3.1. Clinical presentation The purpose of this report is to present clinical, biochemical and genetic information on six patients with Krabbe disease not previous- 3.1.1. Patient 1 ly described in the literature, as well as to assess the sensitivity of Patient 1, the second child born to his parents, was delivered at GALC enzyme analysis in newborn dried blood spots obtained from 37 weeks gestation by emergency cesarean section due to decreased affected individuals. 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 2. Materials and methods formula. Weight and length were below the 5th centile, but followed a parallel curve. Early developmental milestones were normal. At 2.1. Patients 14 months, a clumsy, unsteady gait was noted. Neurological examina- tion demonstrated lower extremity hypertonia and scissoring. EEG Between 2003 and 2008, five patients were diagnosed with and MRI of the brain were reportedly normal. Occupational and phys- Krabbe disease by the Metabolic Division at CHOC Children's (Orange, ical therapy were initiated, however, over the following weeks, he California). An additional patient with late onset disease and pro- stopped walking and lost the ability to sit up or hold up his head. longed survival was referred to the Division during this time. He remained alert and interactive. At 21 months, the patient was admitted for decreased PO intake, swallowing difficulties and con- 2.2. Enzyme analysis cerns for aspiration. Review of systems was significant for two epi- sodes of pneumonia, treated as an outpatient. Physical examination Analysis of GALC activity in leukocytes was performed on all patients revealed relative macrocephaly (weight and length b3rd centile and and on the parents of patients 1, 2 and 5 at the laboratory of David head circumference at the 25th centile), irritability, hypertonia and A. Wenger, PhD, (Lysosomal Diseases Testing Laboratory, Jefferson increased deep tendon reflexes. MRI of the brain demonstrated mark- Medical College, Philadelphia, PA) using the radiolabeled natural edly abnormal T2 prolongation in the periventricular and subcortical 128 R.L. Puckett et al. / Molecular Genetics and Metabolism 105 (2012) 126–131 white matter, as well as in the mid and posterior cerebral hemi- 3.1.3. Patient 3 spheres, the posterior part of the corpus callosum and the pons Patient 3, born at 34 weeks gestation, was the first child for par- (Fig. 1A). There was no enhancement after contrast administration. ents of Mexican descent. Acquisition of milestones within her first A gastrostomy tube was placed due to poor feeding. GALC activity year of life was normal. The patient began walking at 14 months, was found to be low, consistent with Krabbe disease. After discharge, but fell frequently. At 24 months, she manifested generalized tonic– the patient continued to deteriorate and was readmitted for pneumo- clonic seizures and her verbal development stagnated. She began to nia. An EEG demonstrated generalized slowing, but no seizures. He lose vocabulary and, by 30 months, had only two words. Soon there- was, nevertheless, started on phenobarbital, as well as baclofen to after, she became ataxic with prominent toe walking and, by treat his hypertonia. By 25 months, he had lost all previously acquired 36 months, was unable to sit unsupported or walk. When she milestones. He did not smile or interact and had few spontaneous crawled, she often collided with obstacles, as if unable to see them. movements. Hospice care was implemented and the patient died at The diagnosis of Krabbe disease was made by GALC enzyme analysis home shortly thereafter. at 40 months. One month later, she lost the ability to feed by mouth and a gastrostomy tube was placed. By 42 months, she was complete- ly blind and immobile, thereby requiring a wheelchair. Progressive 3.1.2. Patient 2 contractures developed. Between four and 10 years of age, she Patient 2, the second child for parents of Mexican descent, was remained stable with no change in her development. From age 10 born at 32 weeks gestation by cesarean section. Nasogastric feedings to 12, the patient was hospitalized five times due to pneumonia were required for a few days after delivery due to prematurity. She with hypoxia and severe respiratory distress. She recovered with was discharged at four weeks in stable condition. The patient devel- intravenous antibiotics and even required ventilatory support. MRI oped good early head control and sat without support at seven of the brain at 11.4 years demonstrated extensive T2 prolongation months of age, but never crawled. At 11 months, her mother noted of the periventricular and subcortical white matter, thalami, and pos- increased irritability, crying and arching. One month later, she was terior limb of the internal capsule (Figure IB). The basal ganglia, cere- admitted for RSV pneumonia. A gastrostomy tube was placed for bellar hemispheres, and vermis were atrophied. Examination at nutritional support. MRI of the brain demonstrated abnormalities 13.5 years, revealed relative macrocephaly (head circumference at that were suspicious of a leukodystrophy. An EEG was normal. A re- the 50th centile, weight at the 10th centile and length below the peat MRI of the brain was performed at 18 months due to progression 3rd centile), coarse facial features and marked hypertonicity with of symptoms. Findings included a thin corpus callosum, moderate bilateral pes cavus and flexion contractures of the large joints. There global prominence of the cerebral sulci and central ventricular system was exaggerated startle reflex and sustained ankle clonus. Seizures, and abnormal T2 prolongation in the pons, periventricular and sub- characterized only by blinking and lip smacking, occurred daily cortical white matter of the cerebral hemispheres and the cerebellum, despite treatment with phenobarbital and topiramate. The patient consistent with significant white matter disease. By the time of our died at 14.5 years, following admission for respiratory distress. initial evaluation at 19 months, she had lost nearly all of her acquired milestones. Her weight was at the 3rd centile, height was at the 10th centile and head circumference was at the 3rd centile. She was ex- 3.1.4. Patient 4 tremely irritable and hypertonic. She had intermittent tracking and Patient 4, born at full term by vaginal delivery, was the first child startle response to sound. Krabbe disease was confirmed enzymati- for a mother of mixed European ancestry and a father of English, cally. By 23 months, she had myoclonic seizures and no longer dem- French, and Native American descent. Her early development was onstrated visual tracking or response to light. She had four hospital unremarkable. At five months of age, she experienced developmental admissions and several emergency room visits secondary to febrile regression and irritability. Two to three weeks later, she was admitted illness, respiratory distress, seizures and/or . A gastrojejunal to a local hospital for a febrile illness. Work-up demonstrated elevat- tube was placed at 2.5 years due to gastroparesis. By 3 years, she had ed CSF protein. She was treated for gastroesophageal reflux and dis- thermal instability and low muscle tone. Central and obstructive charged home. Her symptoms persisted and, at seven months, she apena followed. She died at home, at 3.5 years of age. was admitted to CHOC Children's. Examination demonstrated relative

Fig. 1. Brain MRI findings in 3 patients with Krabbe disease. A. Patient 1, infantile disease, MRI obtained at 21 m. Marked T2 prolongation is present in the periventricular white matter, subcortical white matter, and the mid and posterior cerebral hemispheres. B. Patient 3, late-onset disease, MRI obtained at 11.4 years. There is extensive white matter T2 prolongation and volume loss. This includes the periventricular and subcortical white matter with secondary enlargement of the ventricles. There is involvement of the thalamus as well as the caudate nucleus. C. Patient 6, infantile disease with hydrocephalus, MRI obtained at 12 m. Severe dilation of the lateral and third ventricles is present. Air is noted in the right frontal horn. There is extensive T2 prolongation in the periventricular white matter. R.L. Puckett et al. / Molecular Genetics and Metabolism 105 (2012) 126–131 129 macrocephaly (height and weight at the 25th centile and head cir- normalized and he regained respiratory effort, he was extubated cumference at the 60th centile) and opisthotonic posturing. MRI of and became slightly more alert. A gastrostomy tube was placed due the brain revealed extensive central and peripheral brain volume to severe and aspiration. The diagnosis of Krabbe disease loss and periventricular deep white matter signal abnormality was made by enzyme analysis, prior to discharge at 12 months. extending from the brainstem and cerebellar hemisphere. The diag- After discharge, he remained minimally interactive and displayed nosis of Krabbe disease was confirmed by GALC enzyme analysis. very little spontaneous movements. Examinations at 13, 15, and She required gastrostomy tube placement at 11 months due to dys- 18 months showed continued macrocephaly, , hypertonia, phagia. At 12.5 months, she had pneumonia requiring mechanical decreased deep tendon reflexes, and sustained ankle clonus. He ventilation. The patient could no longer see or interact with her envi- became progressively unable to manage his oral secretions and ronment and the decision was made to withdraw life support. succumbed to aspiration pneumonia at 21 months.

3.1.5. Patient 5 3.2. Enzyme analysis Patient 5, the second child for parents of Mexican descent, was born at full term by induced vaginal delivery due to decreased amni- The results of GALC enzyme activity in leukocytes are reported in otic fluid. Forceps were used secondary to nuchal cord. There were no Table 1. In each case, the level of enzyme activity was significantly perinatal complications. Developmental milestones were normal decreased, consistent with a diagnosis of Krabbe disease. until six months of age. At that time, the patient also developed irri- tability and incessant crying. She began to receive occupational and 3.3. Analysis of newborn dried blood spots , as well as infant stimulation. At nine months, she was referred to CHOC Children's following a febrile . Loss of The activity of GALC in newborn dried blood spots from all six previously acquired milestones was evident on admission. Physical patients was abnormal by New York State standards (b20% of the examination revealed an alert, well nourished and very irritable daily mean). Results are presented in Table 2. infant with generalized hypertonia and arching. An ophthalmologic exam, including fundoscopy, was normal. Hearing appeared normal. 3.4. Mutation analysis MRI of the brain demonstrated T2 prolongation involving the deep white matter and periventricular region of the cerebral hemispheres, The results of molecular studies on all six patients are summarized cerebellum and brain stem. An EEG was normal. Krabbe disease was in Table 3. Patient 1 was found to be a compound heterozygote for a confirmed enzymatically. A few weeks after the diagnosis was paternally inherited missense mutation, p.Y319C, previously reported made, a gastrostomy tube was placed due to poor PO intake. Her clin- in a patient with infantile Krabbe disease [29], and a maternally ical course worsened progressively over the next two years. She lost inherited missense mutation, p.G41S, described in several late onset all interaction with her surroundings and developed myoclonic sei- patients from Catania [30] Molecular analysis in patient 2 revealed zures and severe , which were treated with levetiracetam, heterozygosity for a novel splice site mutation, c.860+1G>A, and a clonazepam and baclofen. She had ten hospital admissions for . novel missense mutation, c.1865G>T, predicting the substitution of Gastrostomy–jejunostomy tube feedings were required due to severe valine for glycine at position 622 (p.G622V). Patient 3 was found to reflux. Hospice care was arranged and the patient died at home at be heterozygous for a previously unreported deletion of 8 base 33 months of age. pairs, c.602-609delGTCTCCAG, presumed to result in premature ter- mination of protein translation at position 208 (p.G201AfsX8). A sec- 3.1.6. Patient 6 ond pathogenic mutation was not detected, however, two previously Patient 6, born at full term, was the first child for a mother of unreported variants of uncertain significance, c.573+6T>C and Mexican descent and father of Northern European Caucasian descent. c.1623-15C>T, either of which may affect protein splicing, were There were no neonatal complications. At six months, he was devel- identified. The results of molecular studies on Patient 4 demonstrated opmentally normal. Soon thereafter, his development plateaued and compound heterozygosity for a maternally inherited, previously rapidly regressed. At 11 months, he presented to medical attention reported, missense mutation, p.F498S (18) and a novel, paternally with a four day history of lethargy, vomiting, diarrhea, upper airway inherited insertion mutation, c.384_385insA, predicted to result in congestion, difficulty swallowing, and decreased urine output. In the premature termination of protein translation (p.T129NfsX5). Patient emergency department, he was hypothermic (rectal temperature of 5 was found to be heterozygous for the common 30-kb deletion. In 87.8 F), bradycardic (heart rate of 50), and initially hypotensive addition, identified two additional mutations that with a blood pressure of 64/46 mm Hg. After IV hydration, however, have been previously reported on the same allele: a deletion of 17 his blood pressure rose to 135/118 mm Hg, leading to the suspicion nucleotides beginning at position 1822, predicting premature termi- of increased intracranial pressure. A CT scan of the head demonstrat- nation of protein translation (p.T609YfsX7), and a single base change, ed severe tri-ventricular hydrocephalus and diffuse deep white mat- c.574-1G>T, predicting loss of a splice acceptor and subsequent ter abnormalities. He was intubated and subsequently referred to frameshift leading to a premature stop codon (7). Molecular analysis our institution for emergent ventriculostomy. An extraventricular in patient 6 revealed compound heterozygosity for a novel, paternally drain was placed. Postoperative MRI of the brain demonstrated con- tinued ventricular dilatation and extensive T2 prolongation of the periventricular white matter and cerebral peduncles (Fig. 1C). EEG revealed diffuse, disorganized background slowing. A nerve conduc- Table 1 tion study was consistent with peripheral demyelination. The patient GALC enzyme activity in leukocytes (nmol/h/mg protein). remained obtunded without spontaneous respiratory effort for two Patient Patient Mother Father weeks. He could not regulate his body temperature and would become hypothermic without heating blankets. He was sluggishly 1 0.20 2.60 0.80 2 0.06 3.00 2.60 responsive to noxious stimuli, but otherwise displayed no spontane- 3 0.00 N/A N/A ous movements. Examination demonstrated relative macrocephaly 4 0.01 N/A N/A (length at 66th centile, weight at 10th centile, and head circumfer- 5 0.00 1.60 1.30 ence at 87.5th centile), frontal bossing, horizontal nystagmus, 6 0.05 N/A N/A opisthotonus and decreased deep tendon reflexes. As his vital signs N/A = data not available. 130 R.L. Puckett et al. / Molecular Genetics and Metabolism 105 (2012) 126–131

Table 2 considered. Their parents did benefit, however, from the knowledge Retrospective GALC enzyme activity in newborn dried blood spots. of their reproductive risk. Couples who have had a previously affected Patient Sample age Activity % of meana % of daily meanb child can utilize preimplantation genetic diagnosis and/or prenatal (months) (μmol/L h) testing. In addition, transplantation is an option for newborns diag- 1 61 0.12 4.3 3.2 nosed pre-symptomatically due to a previously affected sibling. 2 55 0.19 6.4 4.8 With the promise of treatment for Krabbe disease and other lyso- 3 162 0.43 15.0 11.1 somal storage diseases, newborn screening to permit early diagnosis 4 17 0.28 9.6 7.1 became relevant. Several methods to measure lysosomal enzyme 5 48 0.19 6.4 4.8 6 17 0.28 9.6 7.1 activity in dried blood spots have been described [22,23,34]. Orsini et al. further modified the published methodology to improve the a The mean activity of the 36 specimens from the California Department of Public specificity for the detection of Krabbe disease in the State of New Health was 2.891. b The mean activity from all plates incubated on the same day as the California spec- York [35]. In their publication, 16 Krabbe disease patients and 56 car- imens was 3.91. riers were screened. The positive control samples were obtained from known patients, aged 8 months to 18 years, with the exception of a cord blood sample and a venous blood sample, collected at 7 days of inherited stop mutation, c.441G>A (p.W147X) and a maternally age, on the same patient. The study did not, however, test newborn inherited, missense mutation, c.820C>T (p.R274C). The latter muta- dried blood spots, which had been routinely collected, from known tion has not been previously published, however, it was detected in Krabbe disease patients. combination with the 30 kb deletion in another patient with Krabbe The results of our study support GALC enzyme analysis in new- disease studied by the Neurogenetics Laboratory at New York Univer- born dried blood spots as a sensitive screening test for Krabbe disease. sity. The mutation results in the non-conservative substitution of cys- All six of our patients, including patient 3, who had milder clinical teine for arginine in a highly conserved region. course, were detected by this assay (Table 2). Of note, the activity level for patient 3 would have fallen above the cut-off for follow-up 4. Discussion molecular testing (≤12% of the mean activity) when compared to the mean activity of the 36 California specimens, however, when In this paper, we present six patients with Krabbe disease: three compared to the daily mean of all samples incubated on the same with early infantile disease, one of whom had hydrocephalus, two date, the activity level for patient 3 was below 12% of the mean. Inter- with late infantile onset and one who became symptomatic in child- estingly, the enzyme activity measured by the standard assay for this hood and had a protracted disease course. Patients 1 and 2 presented patient was undetectable (Table 1). Although it is known that enzyme in late infancy (>6 months of age), with subsequent rapid progres- activity does not correlate with disease severity, it would be impor- sion of neurological symptoms and death occurring two to three tant to study additional patients to establish whether enzyme analy- years after diagnosis. Patient 3 presented after two years of seemingly sis performed on NBS cards can detect all patients with mild forms of normal development with acute-onset seizures, verbal stagnation, the disease. In addition to the sensitivity of the assay, our results sup- and loss of ambulation. This was followed by slow disease progres- port the stability of GALC enzyme activity in newborn dried blood sion, spanning more than a decade. Patients 4, 5 and 6 had classic spots that have been stored for years. This is supported by the fact early infantile Krabbe disease, with the course in the latter complicat- that none of the control samples, obtained at the same time and ed by hydrocephalus. stored in the same manner as the patients' samples, were found to To our knowledge, hydrocephalus has only been reported in three have low GALC activity. previous cases of infantile Krabbe disease. In 1974, Laxdal et al. de- With the implementation of newborn screening for Krabbe dis- scribed a patient diagnosed at four months, in whom obstructive hy- ease, comes the difficult task of assessing infants detected by this drocephalus developed over the next six weeks [14]. A second patient method to determine for whom HSCT should be considered. As GALC with infantile Krabbe disease diagnosed at 4 months, developed enzyme activity does not reliably coordinate with disease severity, macrocephaly and tetraventricular hydrocephalus 21 days post bone additional predictors of outcome are desired. Some genotype pheno- marrow transplantation [16]. Recently, Breningstall et al. described type correlations for Krabbe disease are known. With this study, we a third patient, diagnosed at 3 months of age who developed hydro- hoped to contribute to that knowledge. All of our patients were het- cephalus 18 months later [15]. The authors postulated that the erozygous and many carried novel mutations. Six of the GALC muta- change in CSF viscosity caused by elevated protein levels results in tions presented in this report have not been previously described. blockage and subsequent obstructive hydrocephalus. Hydrocephalus These include one splicing mutation (c.860+1G>A), two missense has also been described in other including Alexan- mutations (p.G622V and p.R274C), one deletion (c.602-609del8), der disease, in which the accumulated Rosenthal fibers may lead to one insertion (c.384_385insA) and one stop muation (p.W147X). aqueductal stenosis, [31,32] and in a single patient with adrenoleuko- The large number of novel mutations detected among our patients dystrophy who presented with transient hydrocephalus [33]. provides support for the high incidence of private mutations in the As with most cases of Krabbe disease, our patients were diagnosed GALC gene, limiting the use of genotype to predict outcome in a pre- after the onset of symptoms, too late for treatment with HSCT to be symptomatic infant.

Table 3 GALC molecular analysis.

Patient Onset Mutations Variants of unknown significance Polymorphic background

1 Late infantile p.Y319C p.G41S p.I546T, p.I546T 2 Late infantile c.860 +1G>Aa p.G622Va p.I546T, p.D232N 3 Childhood c.602-609del8a c.573+6T>C c.1623-15C>T p.I546T, p.D232N 4 Early infantile c.384_385insAa p.F498S p.D232N 5 Early infantile c.502T/del c.1822del17b c.574-1G>Tb p.I546T, p.R168C 6 Early infantile/hydrocephalus p.W147Xa p.R274Ca p.I546T

a Previously unreported mutation. b Mutations previously reported on the same allele; parental studies were not available to confirm the configuration for this patient. R.L. Puckett et al. / Molecular Genetics and Metabolism 105 (2012) 126–131 131

Interestingly, only one pathogenic mutation, c.602-609del8, was [10] P. Verdru, M. Lammens, R. Dom, A. Van Elsen, H. Carton, Globoid cell leukodystro- phy: a family with both late-infantile and adult type, 41 (1991) detected in patient 3. It is possible that one or both of the previously 1382–1384. unreported variants that she also carried, c.573+6T>C and c.1623- [11] L. Crome, F. Hanefeld, D. Patrick, J. Wilson, Late onset globoid cell leucodystrophy, 15C>T, are deleterious. Alternatively, one may propose that the Brain 96 (1973) 841–848. [12] E.H. Kolodny, S. Raghavan, W. Krivit, Late-onset Krabbe disease (globoid cell leu- mutation, on a p.I546T, p.D232N polymorphic background, resulted in kodystrophy): clinical and biochemical features of 15 cases, Dev. Neurosci. 13 sufficiently low levels of galactocerebrosidase to cause symptoms. (1991) 232–239. Several GALC gene polymorphisms, including p.I546T and p.D232N, [13] A. Fiumara, L. Pavone, L. Siciliano, A. Tine, E. Parano, G. Innico, Late-onset globoid are known to attenuate GALC activity [4]. These variants are responsible cell leukodystrophy: report on seven new patients, Childs Nerv. Syst. 6 (1990) 194–197. for the overlap in activity that exists between carriers and non-carriers, [14] T. Laxdal, J. Hallgrimsson, Krabbe's globoid cell leucodystrophy with hydroceph- making enzyme analysis unreliable as a method of carrier testing for the alus, Arch. Dis. Child. 49 (1974) 232–235. general population. Interestingly, pathogenic mutations are found on [15] G.N. Breningstall, R.J. Patterson, Acquired obstructive hydrocephalus in globoid- cell leukodystrophy, Pediatr. Neurol. 39 (2008) 279–280. the same allele as polymorphisms more frequently than expected and, [16] M. Caniglia, I. Rana, R.M. Pinto, et al., Allogeneic bone marrow transplantation for for some mutations, may only result in decreased enzyme activity infantile globoid-cell leukodystrophy (Krabbe's disease), Pediatr. 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