Main

The spectrum of phenotypes in Gaucher disease, a common lysosomal storage disorder, is becoming broader with the identification of growing numbers of ethnically diverse affected patients.1 Greater interest in and detection of the disease have been spurred by the advantageous results of regular infusions of mannosyl-terminated glucocerebrosidase (GCase, GBA locus) to supplement the existing mutant GCase in macrophage-derived cells.1,2 The increased frequency of Gaucher disease in the Ashkenazi Jewish population provided the background for initial focus on that group for phenotypic characterization.3 However, more ethnically diverse populations are being defined in the Americas, the Middle East, Africa, and Asia, including China and Japan.3–11 The characteristics of GCase deficiency, hepatosplenomegaly, and bony disease are present in these populations, but the variation in specific organ involvement is great.1 Indeed, outside of the European influence region, Gaucher disease may be predominantly neuronopathic, particularly the subacute or slowly progressing neurologic forms, termed “type 3.”

The classic delineation of the Gaucher disease phenotypes includes types 1, 2, and 3.1 These distinctions are based primarily on the absence (type 1) or presence and severity (types 2 and 3) of central nervous system (CNS) signs.1 Non-neuronopathic Gaucher disease (type 1) is defined by the constellation of visceral signs, the diminished GCase activity in nucleated cells, and the absence of primary CNS involvement. In comparison, types 2 and 3 have CNS signs including variably progressive neurodegeneration, myoclonus and myoclonic seizures, bulbar paresis, and supranuclear gaze paresis.12 Gaucher disease type 2 has been termed “acute neuronopathic” because of the more rapid deterioration of affected patients and an average age of death of 9 months. Gaucher disease type 3, subacute neuronopathic, has a later onset of major CNS signs and a highly variable rate of progression. Some variants have rapidly progressive myoclonic seizures progressing to death in adolescence, whereas other variants manifest only static supranuclear gaze palsy and somewhat diminished cognitive function.

The Swedish Norrbottnian population received initial attention for the delineation of the type 3 variant because of the nearly complete ascertainment and characterization in a group derived from a single founder.13,14 However, neuronopathic variants of Gaucher disease in this Swedish population span a spectrum from very severe, early infantile disease, similar to type 2 or acute neuronopathic disease, to a more subacute, slowly progressive, late-onset variant (type 3) with myoclonic seizures and diminished intelligence. In addition, kyphoscoliosis is a predominant skeletal manifestation.13,14 Even within this population, with an apparently homogenous GBA genotype (L444P/L444P), the variation in intelligence outcome and other neurologic signs and symptoms is broad among and between families.13 This variation in neuronopathic phenotype and skeletal disease also is apparent in other populations, including those from Japan and the developing populations in South America, Central America, and Eygpt.15–18

The Polish Gaucher disease population presents a unique ethnic population since the catastrophe of the Holocaust nearly eliminated the Ashkenazi Jews from this Eastern European population. This tragedy and centuries of relative isolation of the Polish Jewish population within ghettos dramatically influenced the ethnic composition of Gaucher disease in Poland so that the predominate non-neuronopathic variant and the associated genotype (i.e., N370S) among the Ashkenazi Jews are not prevalent among ethnic Poles. In this report, a characterization of the neuronopathic variation is provided for subacute neuronopathic Gaucher disease among the ethnic Poles as well as full genotype characterization of these patients with genotype/phenotype correlations.

MATERIALS AND METHODS

Patient assessment

Patients were ascertained by referral to A. Tylki-Szymañska at the Children's Memorial Health Institute in Warsaw, Poland, a central referral unit in Poland. All patients were evaluated by A. Tylki-Szymañska with physical and neurologic examinations on an annual basis. The intelligent quotient assessments were performed in all patients using the Wechsler psychometric test every 2 years. All patients were confirmed by deficient GCase activity in peripheral blood leukocytes or cultured skin fibroblasts.

DNA extraction

Peripheral blood samples were collected and stored at room temperature. DNA was extracted within 1 week of collection using the PureGene kit from Gentra Systems (Minneapolis, MN). DNA concentration was normalized to 50 ng/μL in 10 mM Tris pH 8 and stored frozen until analysis.

GBA sequencing

Long-range polymerase chain reaction (PCR) was used to amplify the complete GBA gene in a single 7269-base pair fragment. To prevent the coamplification of the GBA pseudogene (pGBA), the forward primer was designed from an area with no sequence homology between GBA and pGBA (forward primer: TTC TCC ATG CAA ATC TGT GT; reverse primer: GAA CCA GAT CCT ATC TGT GC). Genomic DNA (100 ng) and 10 pmol of each primer were used with 1 unit of enzyme mixture in buffer system number 2 from the Expand Long Template PCR Kit (Roche Diagnostics Corporation, Nutley, NJ). Replicate (4 × 25 mL) reactions were generated to produce enough template for the sequencing reactions. The annealing temperature was 60°C. Extension time was 280 seconds for the first 10 cycles and was increased by 20 seconds per cycle for an additional 20 cycles for a total of 30 cycles. Long-range PCR products from the four replicates were pooled and purified using the Qiaquick PCR cleanup kit (Qiagen, Venlo, The Netherlands). This approach may miss large (whole gene) deletions (long range) and smaller deletions and mismatches (short amplicons) that exist within a primer site (i.e., no amplification). However, because only a single large deletion complete gene has been reported,19 the likelihood of missing an allele by the above method is small.

Sequencing reactions were performed by the CCHMC DNA Sequencing Facility using an ABI 3730xl DNA Analyzer (Applied Biosystems, Foster City, CA). A total of 22 fragments with overlapping ends were used to cover the complete GBA intronic and exonic sequences.

Sequence files were called using the Phred program, and quality scores were generated for each base. The 22 sequence reads were assembled and aligned to the reference GBA sequence (GenBank accession J03059) using Sequencher v4.1.2 (Ann Arbor, MI). Every matching base call with a Phred quality score less than 15 were automatically color coded and manually checked for the presence of a variation.

Haplotype/allele assignment

Long PCR products were T-A cloned into the TOPO-XL system (Invitrogen, Carlsbad, CA) to assign multiple heterozygote mutations to their respective alleles. Eight colonies were picked from each individual and individually genotyped.

A 10-allele probe set was developed for the SnaPshot multiplex genotyping system (Applied Biosystems) that can simultaneously determine the genotype for the following coding and intronic GBA mutations, using the long-range PCR product as a template: 3002G>A, 3715G>C, 4179A>G, 6021A>C, 7031A>G, IVS2, E326K, D409H, L444P, and R463C. In the case of individuals presenting mutations not included in this set, the clones were resequenced using a primer that would read across the mutation site. Variations from the reference sequence (no. J03059) are presented in Table 3. This reference sequence is used because it is identical to the majority (60%; 94/158) of the alleles characterized in this laboratory (unpublished observation), excluding known exonic point mutations, and contains the majority of recognized common non-Gaucher disease-related sequence variants. The convention of displaying the predicted amino acid substitution is used (e.g., L444P and R463C). When presented without additional annotation, identity to all other nucleotides in J03059 is implied. When a nonsynonymous point substitution occurs within the context of intronic polymorphic variation (i.e., different haplotypes), the variations are indicated as transitions or transversions from J03059. The reference sequence, J03059, is based on the original sequence reported, as modified and updated as of November 4, 2004.

Table 3 Phenotype/genotype findings among ethnic Poles with Gaucher disease type 3
Full size table

PATIENT CHARACTERISTICS

Mutation analysis of GBA in the total population with Polish Gaucher disease shows allele frequencies of alleles containing N370S or L444P mutations of 23% or 50%, respectively. The unidentified mutations made up 7.6% (Table 1). This distribution differed significantly from that in the neighboring Czech and Slovak populations in whom N370S and L444P account for 48% and 27.5% of mutant alleles, respectively.20 In a non-Jewish British population, of 54 screened alleles for these two mutations, 26% were N370S and 35% were L444P.21

Table 1 Allele distributions in patients with Polish Gaucher disease
Full size table

Among patients with Polish Gaucher disease, the most frequent genotype was L444P/L444P (16/47) (Table 2). In the group of patients homozygous for alleles bearing L444P, 13 of 16 have a phenotype resembling that of Norrbottnian lineage patients (see below).

Table 2 Genotype frequencies in the patients with Polish Gaucher disease
Full size table

Type 3 patients account for approximately 40% of the population with Gaucher disease in Poland. The phenotypes of these patients can be delineated into two groups, a group of three patients (Table 3, patients 14, 15, and 20) with mild hepatosplenomegaly, and earlier development of neurologic signs, including strabismus, myoclonus, epilepsy, supranuclear gaze paresis, a characteristic mask-like facial expression (Fig.1), and, occasionally, retroflexion neck. Development quotient and intelligence quotient (IQ) are usually within normal limits. These patients have outbursts of anger and irritability that occur periodically. The above group is similar to that described as type 3a.22

Fig. 1
figure 1

Four patients with Gaucher disease type 3a with pronounced neurologic signs, including a characteristic mask-like face, strabismus, supranuclear gaze palsy, and poor upward gaze initiation. Patients 15 and 16 are siblings and display phenotypic variation within a family. Patient 16 has more severe CNS, skeletal, and lung disease than his sister. Both siblings have moderate hepatic and splenic involvement. Patient 10 was splenectomized at 18 months for massive splenomegaly. The genotypes differ only by a single unique, intronic base substitution at position 5332 (C>T) on one L444P allele. Also, one L444P allele from patient 20 has reference sequence identity, except at codon 444.

Full size image

The larger type 3 group (13 individuals) developed neurologic signs later. Supranuclear gaze paresis usually predominates; massive hepatosplenomegaly, wasting, and progressive skeletal abnormalities are characteristic. Bone pain and crises have not occurred. Generalized osteopenia occurs in the spine and leads to severe thoracic kyphosis (Fig. 1). The lack of neurologic manifestations in childhood often leads to classification of type 1. The details of individual patients are summarized in Table 3. In 22 patients classified as type 3, there were 19 genotypes.

These patients with Polish Gaucher disease type 3 are characterized by early onset (first year) of massive hepatosplenomegaly (Fig. 2). In almost all untreated patients, massive splenomegaly necessitated splenectomy before 3 years. Psychomotor development is usually age-appropriate, but physical development was very poor. Subtle neurologic signs, that is, supranuclear gaze palsy and a mask-like face, generally appear before 4 to 5 years with subsequent characteristic thrusting head movements. In the third decade affected patients can present with myoclonus and scanning speech. From 5 to 6 years splenectomized patients (no enzyme replacement therapy) experience progressive thoracic kyphosis, chest deformation (barrel chest), and sternal protrusion (Fig. 3). Bone crises and significant bone pain are not a component of the phenotype. These characteristics are very similar to those described in the “Norrbottnian phenotype” from Sweden.13,14

Fig. 2
figure 2

Patients with Gaucher disease type 2b and mild neuronopathic and variable visceral disease. Patients 21, 12, and 7 were aged 4, 12, and 8 years, respectively. Patients 21 and 12 shared one L444P encoding allele. Patient 7 was homozygous for L444P encoding alleles that otherwise did not differ from the reference sequence.

Full size image
Fig. 3
figure 3

Progressive “Norrbottnian like” phenotype in a male patient who was splenectomized at approximately 2 years. Characteristic initial and progressively severe kyphosis is present. The patient also has supranuclear gaze palsy and a mask-like facies as the predominant neurologic manifestation beginning at approximately 5 years of age. The patient's age in the various images (inset numbers); frontal view at age 27 years (small inset). He is homozygous for an L444P encoding alleles that has several intronic polymorphic changes.

Full size image

GENOTYPES

The GBA alleles were sequenced from 22 patients with Gaucher disease type 3 who were ethnic Poles. A total of 15 individual mutations were observed in the cohort of 22 patients and were distributed among nine different haplotypes. All but two patients had at least one L444P-bearing allele. Three different haplotypes containing L444P were observed and characterized by intronic polymorphic variations. These polymorphic variants have been observed with frequencies of 8.1% and 9.1% in our population of 158 individuals having alleles bearing other Gaucher disease mutations. The haplotypes for each patient are shown in Table 3. The indicated base changes in the alleles are shown as differences from the reference wild-type GBA. Three previously unreported sequence variations were found, one intronic (5332C>T) (the sib-pair 15 and 16, and case 22), one encoding for R433S (case 3), and an allele encoding V305L (case 22). Patient 8 had two point substitutions that predicted amino acid changes, E326K and A446P. The E326K is a polymorphic variant that has minor effects on enzyme activity.23 The A446P mutation profoundly decreases GCase function (B. Liou, MS and G.A. Grabowski, MD, unpublished data, 2005). The phase of each allele was assigned unambiguously by cloning the long-range GBA PCR product and individually genotyping each clone.

DISCUSSION

The phenotypes of Gaucher disease type 3 in ethnic Poles are similar to that described in other populations. Specifically, there seems to be two major expressions of this phenotype; one with major visceral involvement and a second with late-onset progressive myoclonic seizures from progressive CNS disease. This segregation of phenotypes22 serves a useful purpose in descriptions of such phenotypes throughout the world, but a continuum of neurologic phenotypes populate the spectrum between these descriptors.12 In the ethnic Polish population, the skeletal involvement in patients with severe visceral disease has greater similarity to that described in the Swedish population than in other groups. The severe kyphoscoliosis has not been extensively reported in populations other than the Poles and Swedes. This has not been a reported feature of the type 3 variants in the Japanese or Egyptian populations, but with the broader description of neuronopathic variants in South America and elsewhere, this may be a more common feature than previously appreciated. Also, bone pain and crises are rare among these patients. One-third of the ethnic Poles exhibiting neuronopathic manifestations were adults at the time of last examination and showed relatively mild cognitive defects as measured by the Wechsler IQ scale. This is also similar to the characterization of the Norrbottnian-derived Swedish Gaucher population.13,24 In these reports, even in siblings with substantial visceral variation, some, but not profound, mental retardation was a component of the disease. This variation reemphasizes the continuum of phenotypic variation among variants of the lysosomal storages diseases in general25 and Gaucher disease in particular.12 The presence of a mask-like facies in many of the Polish patients may represent basal ganglia involvement similar to that suggested in Parkinson's variants in adults.26

A broad similarity in kyphoscoliosis and lesser IQ involvement, and a commonality of the L444P/L444P genotype exists between the ethnic Poles and Swedes. Indeed, the Polish and Swedish histories have been intertwined for centuries. In the second half of the 17th century Sweden invaded Poland with such massive numbers of troops that this period is known as the “Swedish Flood.” One wonders whether the complete GBA sequences in these populations would reveal similarities in the L444P encoding alleles. Indeed, the only evidence available indicates that a patient (case 22) of partial Swedish descent had a L444P allele containing polymorphic variants common in this Polish population. It would be interesting whether this L444P allele predominated in the Swedish Gaucher disease type 3 population.

Since 1973, Gaucher disease has been diagnosed enzymatically in Poland. Over the past 27 years, 56 patients from 48 families with either type 1 (non-neuronopathic) or type 3 (subacute neuronopathic) diseases have been identified.27 On the basis of a population of approximately 40 million, this would give a frequency of approximately 1 in 700,000 in that population. This lower frequency may be the result of underdiagnosis and underrecognition of the disease; the availability of enzyme therapy since 1995 led to a nationwide educational campaign among doctors to search for patients with Gaucher disease. However, there has not been a concomitant significant increase in the number of diagnosed patients.

Thus, for historical reasons related to World War II and the Holocaust, Gaucher disease may have a lower frequency in Poland than in other eastern European countries. Poland had more than 95% of ethnic Poles after World War II. Furthermore, as a result of World War II, approximately 50% of Poland's eastern territory was lost and approximately 35% of territory was added in the West. This eastern component was returned to Poland after the war and settled mainly by ethnic Poles expelled from eastern territories and other parts of the country. This led to an admixture population in the western territories, but the southeastern region of Poland, where many of the previously expelled Poles settled, is the primary area of concentration for Gaucher disease. Consistent with these observations, Gaucher disease type 1 is underrepresented in Poland, relative to other European countries (sans Sweden) because these patients account for only approximately half of the Gaucher disease cases. Even among this type 1 population, in which the N370S allele predominates in other European countries, N370S homozygosity was detected in only one Polish patient.

The distribution of specific alleles containing the L444P mutation is similar to that in other populations. The intronic polymorphic variation is similar to those reported in the reference sequence (GenBank J03059). However, a new intronic variation, a nucleotide 5332C→T transition, was found in three patients, none of whom were homozygous. Thus, in the population of L444P-containing genomes, three different L444P alleles were discovered including the most predominate consensus wild-type sequence. A new mutation encoding a serine substitution for an arginine at amino acid 433 was discovered in a homozygous state suggesting consanguinity. Careful clinical follow-up over the next several years with this patient will be important to more fully characterize the phenotype associated with this rare or unique genotype. In addition, the R463C mutation was found in one patient with low normal IQ. At 8 years of age, supranuclear gaze palsy was not present even though the other allele contained an IVS2 mutation, that is, a null allele. It is unclear in this family whether the low normal IQ is related to Gaucher disease because other siblings without Gaucher disease also were slightly retarded. Finally, the E326K polymorphic variant was found in association with the A446P allele that could have arisen from the pseudogene by conversion. The E326K, at least in some families, seems to be a nondisease polymorphic variant,23 whereas the A446P leads to a severely disrupted GCase enzyme. This could explain the combination of the supranuclear gaze palsy and skeletal abnormalities with the L444P allele in case 8.

The numbers of patients involved are not significant enough to draw conclusive genotype and phenotype correlations. However, the neuronopathic phenotype is relatively mild and consistent with prolonged survival. This indicates the appropriateness of enzyme therapy for management of their severe visceral disease.

It is clear with continued complete gene sequencing in Gaucher disease that the relationships of specific allele variants with genotypes may become more apparent. Whether strict correlations will be discerned or only general conclusions can be refined may come only after detailed phenotyping of dozens of patients. In addition, the onset of early severe visceral disease and the institution of enzyme therapy may alter some of the neuronopathic manifestations,30 as well as the skeletal course, and thus the “natural” history of this disease and other lysosomal storage diseases now being treated may be irrevocably altered and irretrievable.