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Файл:Ординатура / Хирургия / Библиотека им академика М.И. Перельмана / Книга_2984_Библиотеки_им_академика_М_И_Перельмана
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S. Sundaram, S. V. Chandrasekharan and S. S. Nair
222
Because urine sulfatide excretion is highly variable, ideally it is measured
in a 24-hour urine sample or normalized to the urinary excretion of
creatinine. Urinary sulfatide excretion is increased in patients with MLD
and is more than ten times the normal value. It is only elevated by less than
ten times the normal value in patients with saposin B deficiency. Urinary
sulfatide excretion is normal in patients with pseudo deficiency [4]. If ASA
enzyme levels are found to be low, demonstration of urine metachromatic
granules can differentiate pseudo deficiency state from MLD [27].
Multiple sulfatase deficiency can also have an elevated urine sulfatide
excretion and low ASA enzyme activity. However, the presence of
dysmorphic features, dysostosis multiplex and ichthyosis may favor a
diagnosis of multiple sulfatase deficiency rather than MLD [71].
8.5. Electroneurography (Nerve Conduction Study)
Peripheral neuropathy is constantly present in early onset forms of
MLD in whom it manifests as a demyelinating neuropathy.
Electroneurography shows progressive and severe reduction of nerve
conduction velocities [46]. In a review of 12 patients, it was found that
infantile form was associated with a severe diffuse demyelinating
sensorimotor peripheral neuropathy with conduction velocities in the range
of 19–21 m/s in median nerve and 15–20 m/s in tibial nerve [5].
Conduction blocks in multiple nerves were also reported in MLD which
can be confused with an acquired demyelinating polyradiculoneuropathy if
the patient has a rapid progression [5, 46]. Conduction blocks are typically
associated with acquired form of demyelinating neuropathies such as
Guillain Barre syndrome and chronic inflammatory demyelinating
polyneuropathy. But many exceptions to this rule have been reported in
some hereditary neuropathies like Charcot Marie Tooth disease, MLD, and
Krabbe disease which infrequently demonstrate conduction block [72].
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8.6. Nerve Biopsy
The utility of nerve biopsy for diagnosis of MLD is currently limited
with the wide availability of molecular genetic test. When an alternate
diagnosis is to be ruled out, sural nerve is the preferred biopsy site. The
pathognomonic finding is the presence of metachromatic granules with
loss of large myelinated fibers and presence of thinly myelinated fibers
(Figure 8).
Figure 8. Histopathology of nerve in MLD: Cresyl violet stain A) showing perivascular
brown metachromatic deposits (arrow) within the endoneurium. B) Semithin section
stained with Toluidine blue showing myelin folds (arrows) along with several thinly
myelinated fibers (arrow heads). (Courtesy: Dr. Anita Mahadevan and Dr. Radhika
Mhare, Department of Neuropathology, National Institute of Mental Health and
Neurosciences, India).
8.7. Other Tests
Cerebrospinal fluid analysis shows elevated protein levels especially in
childhood forms, but this is not a uniform finding in adult forms. Routine
hematological and biochemical parameters are usually normal and not
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S. Sundaram, S. V. Chandrasekharan and S. S. Nair
224
helpful for the diagnosis in MLD [40]. Screening of patients for gall
bladder pathology can detect gall bladder distension and polyps. In a study
of 34 MLD patients, almost one fourth had gall bladder polyps and one-
third underwent cholecystectomy. Normal gall bladder was seen only in
23% of patients. Histopathology revealed abnormal findings such as
hyperplastic polyps, metaplasia and sulfatide storage [73].
8.8. Genetic Tests
ARSA gene analysis provides the most important diagnostic tool for
MLD. As described previously, the four most common mutations
(c.459+1G > A, c.1204+1G > A, p.Pro426Leu, and p.Ile179Ser) accounts
for one half of the cases. Further sequencing of the entire coding region of
ARSA has often led to identification of novel variants. During the
interpretation of the tests results, it is necessary to differentiate the
pathogenic variants (ARSA-MLD) from the pseudo-deficient alleles
(ARSA-PD).
The molecular genetic testing approaches can include a targeted single-
gene testing, i.e., sequence analysis of ARSA gene alone or a targeted
multi-gene panel for leukodystrophy. If the clinician is definitive about the
diagnosis of MLD after clinical and radiological concordance along with
low residual enzymatic activity, single gene sequencing of ARSA is the test
of choice. Since Sanger sequencing of a single gene is cost effective, in
situations where clinical suspicion is high, this approach can be pursued. If
only one or no pathogenic variant is found, gene-targeted deletion/
duplication analysis should be performed next. The multigene panel for
leukodystrophy including PSAP sequencing is pursued later if broader
diagnostic entities need to be excluded. If the above tests fail to yield
results, a comprehensive genomic testing such as whole exome sequencing
and genome sequencing can be considered in a clinical or research setting
[4].
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Table 2. Differential diagnosis for late infantile MLD
Disorder
Genetics
Protein
deficient
Clinical Features
MRI
Diagnosis
Multiple Sulfatase
deficiency
SUMF1
Formyl
glycine-generating
enzyme (FGE) deficiency
Gross motor and speech
delay, coarse facial
features, deafness, HSM,
kyphoscoliosis, icthyosis
Progressive demyelination,
prominence of the
perivascular spaces, cerebral
volume loss, hydrocephalus
Decreased activity of at least
two sulfatase enzymes on
lysosomal enzyme testing
analysis, elevated urinary
GAG levels, elevated urinary
sulfatides
ALD
(cerebral form)
ABCD1
Peroxisomal membrane
protein ALDP
(transmembrane transport
of VLCFA)
Cognitive decline, apraxia,
astereognosis, auditory
impairment, decreased
visual acuity, hemiparesis
or spastic tetra paresis,
cerebellar ataxia and
seizures
Predominant parieto-
occipital white matter and
corpus callosal involvement,
Schaumberg's zone and
contrast enhancement
Genetic tests and elevated
VLCFA levels
Infantile Krabbe
disease
GALC
Galactocerebr-oside-β-
galactosidase
Loss of motor milestones,
spasticity, truncal
hypotonia, OA esotropia,
seizures, painful tonic
spasms, demyelinating
peripheral neuropathy
Demyelinating pattern with
involvement of
periventricular white matter
and centrum semiovale,
bilateral T2 hypo intensity of
thalamus, optic nerve and
chiasm involvement
Enzyme assay and genetic
tests
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S. Sundaram, S. V. Chandrasekharan and S. S. Nair
226
The genetic testing needs to be done after providing detailed genetic
counselling and obtaining a written informed consent from the patient or
parents in case of minors. Genetic testing of the unaffected siblings,
parents and other family members needs to be contemplated in consultation
with clinical geneticist and considering the individual case scenario.
Screening of the unaffected siblings is warranted, as HSCT has been
proven to be more effective in pre-symptomatic and early stages of the
disease process. Genetic testing is usually performed from samples from
amniocentesis or chorionic villous biopsy for prenatal diagnosis [40].
Hence, optimal genetic diagnosis and prenatal counselling require a
multidisciplinary consultation involving neurologist, geneticist and
obstetrician.
Table 3. Selected differential diagnosis of late onset juvenile
and adult forms of MLD
Conditions
Disorders
Lysosomal storage
disorders
Adrenoleukodystrophy, Krabbe disease, Neuronal ceroid
lipofuscinosis (Kuf’s disease), Niemann-Pick disease type C,
Gaucher's disease (type 2 and 3), GM2 gangliosidosis
Other metabolic disorders
Fahr’s disease, MELAS, other mitochondriopathies, Cerebro-
tendinous xanthomatosis, Wilson’s disease,
Methylenetetrahydrofolate reductase deficiency, Homocystinuria
Leukodystrophy
Alexander disease, Vanishing white matter disorder, Adult
polyglucosan body disease, Hereditary diffuse leukoencephalopathy
with axonal spheroids (HDLS)
Early onset forms of
neurodegenerative
dementia
Alzheimer dementia, Frontotemporal dementia, Lewy body dementia
Vascular dementia
Small vessel ischemic disease, CADASIL, cerebral amyloid
angiopathy, CNS vasculitis
Heredodegenerative
dementia
Huntington’s disease, Spinocerebellar ataxia
Infections
Creutzfeldt Jakob disease, Subacute sclerosing pan encephalitis,
HIV, Neurosyphilis, Whipple disease, PML
Inflammatory disorders
Multiple sclerosis, Sarcoidosis
Nutritional
Vitamin B12 deficiency, Pellagra, Thiamine deficiency
Endocrinological
dysfunction
Hypothyroidism, hypo and hyperparathyroidism
Psychiatric disorders
Schizophrenia and other psychosis, bipolar disorders
Miscellaneous
Heavy metal poisoning like mercury, arsenic and lead
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9. DIFFERENTIAL DIAGNOSIS
Several disorders need to be enlisted as alternate possibilities while
dealing with a phenotype similar to MLD. Multiple sulfatase deficiency
(Austin disease), Krabbe disease and adrenoleukodystrophy are a few
differentials which need to be excluded in late infantile and early juvenile
forms of MLD (Table 2) [27]. There is a broader differential diagnosis for
late onset forms and these cases should be approached like any young
onset dementia. Table 3 provides a concise list of the major differential
diagnosis [74]. An optimal combination of clinical evaluation, MRI pattern
recognition, biochemical analysis and molecular genetic tests can help in
the accurate diagnosis of MLD.
10. THERAPEUTIC APPROACHES IN MLD
Therapies for MLD can be broadly classified into disease-specific
therapies and general therapies.
10.1. Disease Specific Therapies
Specific therapies for MLD target the rectification of enzyme
deficiency and/or sulfatide toxicity using cell-based or protein-based
methods. None of the current therapies are curative, but many holds
promise for the future. Allogenic HSCT, enzyme replacement therapy
(ERT) and gene therapy are the three main avenues of therapy widely
explored in animal models and human studies. Small molecule-based
therapies for substrate reduction and enhancing enzyme levels also warrant
more biochemical and therapeutic trials in MLD. (Figure 9). Among these,
only HSCT is available for clinical use at present while others are still in
the experimental phases [75].
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S. Sundaram, S. V. Chandrasekharan and S. S. Nair
228
Figure 9. Pharmacological strategies in MLD. Mutations in the ARSA resulting
misfolded or unfolded proteins, which are degraded by lysosomal hydrolases,
endoplasmic reticulum associated degradation or ubiquitin proteosomal system.
10.2. Concept of Replacement Therapies in MLD
The most promising therapies in MLD attempt the normalization of
ASA levels in brain and CSF by HSCT, gene therapy or ERT. In humans,
about 10-15% of normal ASA activity is adequate to prevent
manifestations of MLD, a principle employed in most of the replacement
strategies [4]. The concept of HSCT (allogenic and gene-corrected
autologous) arose from the principle of ‘cross-correction’ noted in the late
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1960s and 1970s. When cells of patients with LSD, deficient in specific
enzymes, were exposed to media containing cells with normal enzyme
activity, the metabolic defect was corrected by enzyme transfer from the
normal to the deficient cells [76]. In MLD, this is achieved by M6P
residues in the ASA which are recognition markers for endocytosis of the
enzyme. During biosynthesis of ASA, a considerable fraction of the
enzyme escapes to the extracellular space which is reabsorbed into the cell
through M6P receptors on the plasma membrane and transported back to
the lysosomes. The same mechanism facilitates the cross-correction of
enzyme deficient cells from the metabolically normal cells. Intravenous
and intrathecal ERT also rely on the endocytosis of ASA by M6P or
mannose receptors [77].
10.3. Hematopoietic Stem Cell Therapy
The reassuring success of HSCT in early Hurler syndrome paved the
way for the similar trials in other inborn errors of metabolism (IEM). The
principle of ‘cross correction’ is employed here with the premise that toxic
substrate accumulation can be prevented by the presence of newly
transplanted cells capable of supplementing the deficient enzyme [76]. The
donor-derived hematopoietic cells secrete ASA into the blood, but the
enzyme is incapable of crossing the blood brain barrier (BBB). However,
the transplanted cells can migrate to the CNS compartment and
differentiate into microglial cells which act as the source of the deficient
enzyme in the brain (Figure 10) [78].
HSCT for IEM uses allogenic hematopoietic cells from the bone
marrow, peripheral blood (mobilized by growth factors), and umbilical
cord blood. The donor cells from a non-diseased volunteer are prepared in
vitro and infused into the recipient who then undergoes an intensive
immunosuppressive regimen to reduce likelihood of graft rejection and
graft versus host disease. The donor-derived stem cells or progenitor cells
cross the BBB, engraft within the CNS and differentiate into cells of
myeloid lineage and microglial cells capable of secreting ASA [79]. In
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S. Sundaram, S. V. Chandrasekharan and S. S. Nair
230
addition, the myeloid cells play an important role in limiting the
pathogenic processes contributing to tissue damage including inflammation
and oxidative stress.
Figure 10. Hematopoietic stem cell transplantation: Allogenic hematopoietic cells from
the bone marrow, peripheral blood and umbilical cord blood is used for hematopoietic
stem cell transplantation (HSCT). The donor cells from a healthy volunteer was
prepared in vitro and infused into patient. The donor-derived stem cells or progenitor
cells differentiate into different blood cell lineages. Myeloid precursors differentiate
into monocytes and cross the BBB. They finally differentiate into microglial cells and
recreate arylsulfatase-A enzyme.
The expertise for allogenic HSCT is widely available and has been
attempted in more than 100 subjects with MLD across different centers.
The technique can potentially halt the progression of the disease, especially
in the early and pre-symptomatic stages. The main limitation is the
prolonged time to disease stabilization due to the slow repopulation of the
cells, spanning a period of 12 to 24 months [80]. HSCT is generally
ineffective in late infantile MLD and MLD with severe neurological
symptoms where the speed of disease worsening overwhelms the
correction of enzyme level. Hence, the outcome is better when HSCT is
given early in the disease course (pre-symptomatic or mild symptoms) and
when the symptom onset is later in life (adult onset MLD than late infantile
or juvenile). In murine models, transplantation of hematopoietic stem cells
within the cerebral lateral ventricles resulted in faster engraftment of
microglia-like cells [81]. A second limitation is that the improvement is
confined to the CNS compartment whereas no effect is noted for peripheral
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neuropathy. Significant gait deterioration with loss of ambulation can
occur due to worsening peripheral neuropathy over time [82]. High risk of
transplant related complications and mortality also dampen the desirability
of the technique. Graft versus host disease, graft failure and infections
contribute to mortality in 10 to 15% of subjects, although the incidence of
these have markedly reduced over the decades due to improvement in
procedures and immunosuppressive regimens [75].
In the largest single center experience with HSCT, among 40
transplanted patients with MLD, the estimated 5-year survival rate was
59%. Most of the deaths were related to transplant related complications.
Juvenile and adult onset MLD had better long-term outcome with
stabilization of MRI. The source of stem cells was associated with the
outcome. Bone marrow from sibling and umbilical cord blood
transplantation from unrelated donors showed better outcome compared to
bone marrow from unrelated donors [83]. In comparison with non-
transplanted patients, transplant recipients had better language and motor
functions on long term follow up. Stabilization of motor functions occurred
in 55 to 60% the transplanted subjects [84].
MRI stabilization is noted in transplant recipients pari passu with the
clinical outcomes [75]. Favorable baseline MRI measures for a good
outcome included low MRI-MLD severity score (especially temporal and
parieto-occipital sub scores) and lack of U fibers involvement [83]. Case
series and reports have shown increase in white matter lesions for 6 to 12
months after transplant indicating the delay in achievement of enzyme
activity. A reversal of high choline - NAA ratio in MRS was reported after
12 months, indirectly indicating the time to correction [85-87].
The good prognostic categories for HSCT in MLD are therefore, those
with mild disease (mild motor and cognitive dysfunction), later age of
symptom onset and lower MRI severity scores. The worst prognostic
categories include severe neurological symptoms and prolonged duration
from symptom onset to institution of therapy. The current recommendation
for HSCT in MLD is to offer this as a standard for adult MLD in the early
or asymptomatic phases and as an option for late infantile and juvenile
MLD [88].
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