What is a modern diagnostic approach?

A simplified and streamlined diagnostic protocol can be downloaded here. NCL diagnostic protocol (pdf file). For more detail consult the separate page containing diagnostic algorithms or an expert in NCL clincal features and diagnosis.

Enzyme analysis

It is now possible to measure the enzyme activities of the CLN1/PPT1, CLN2/TPPI and CLN10/CTSD enzymes. Low levels are definitive of NCL caused by mutations in these genes, even in cases of adult onset. These enzyme tests can be carried out using blood, skin biopsy or saliva as the source of cells and care must be taken to use the most specific assays possible, especially for cases of later onset. Nowadays it should be a routine matter to perform these enzyme assays on all cases of NCL, whatever the age of onset, to confirm NCL caused by mutations in these genes. However, cases with apparently normal enzyme levels in blood samples but clinical features consistent with disease caused by mutations in CLN1/PPT1, CLN2/TPP1 or CLN10/CTSD should be followed up with enzyme and EM analysis of fibroblasts obtained via skin biopsy.

Mutation analysis

The identification of a mutation in both copies of an NCL gene is definitive for diagnosis of NCL and of NCL type. This also allows accurate carrier and prenatal testing. Although the enzyme tests for CLN1/PPT1, CLN2/TPPI and CLN10/CTSD are confirmatory for diagnosis if mutations are present in either of these genes, mutation analysis of CLN1/PPT1, CLN2/TPP1 or CLN10/CTSD is routinely performed since knowledge of the underlying mutations is usually required. Defining the mutations present in cases of variant LINCL is at present the only way to reliably assign NCL type. For some patients no mutations in any known NCL gene will be found, either because they carry mutations in a gene that has not yet been identified, or because their mutations are undetectable by the techologies used (eg they may be in the gene promoter or intronic regions), or because they do not have NCL. If a mutation in a novel NCL gene is suspected, perhaps because the disease progression is unusual, it is recommended that the contribution of samples for research is considered to aid in future gene identification.

Electron microscopy

Cells from patients with NCL accumulate storage material which can be examined ultrastructurally using electron microscopy. Various patterns of these NCL-specific structures are recognised. Diagnosis, particularly of variant types, can therefore be aided by electron microscopy. The tissue best suited to this is a skin biopsy. A 3 mm punch biopsy deep enough to include sweat glands is ideal. Skin contains a wealth of diverse types and therefore provides a higher diagnostic yield than lymphocytes. The most important cell type in the skin for diagnosis is the secretory eccrine sweat gland epithelial cell. It is good practice to require storage in at least two cell types to reach a diagnosis of NCL. Fibroblast cells in general do not exhibit evidence of storage, except for GRODs arising from mutations in CLN1. Granules from mast cells are a frequent source of misdiagnosis. The biopsy should be taken from a site where sweat glands are present (eg the inside of the upper arm but not the axillary region, or the forearm) and ideally the biopsy should be transferred to 2.5% buffered glutaraldehyde for preservation or if this is not practical 10% buffered formaldehyde. Alternatively examination of circulating lymphocytes may also be of use and is minimally invasive. Lymphocytes should be isolated as a buffy coat and well fixed. Care must be taken to distinguish between NCL-specific material and normally ocurring similar structures, particularly after a viral illness. Use of peripheral blood alllows reliable and specific diagnosis of NCL and can readily distinguish between INCL, classic LINCL, JNCL and variant NCL types. In the past rectal biopsy and tissues such as conjuntiva or skeletal muscle were used but this is not so common now, and inclusions may take on a different pattern in muscle fibes complicating diagnosis of NCL type. For diagnosis of adult onset NCL, a brain biopsy might be considered since storage may not be found in a skin biopsy or circulating lymphocytes. For a brain biopsy, a small piece should be snap frozen in liquid nitrogen, and the main portion divided for routine analysis (preserved in buffered formaldehyde) and ultrastructural investigation (buffered glutaraldehyde). This will provide ample opportunity to achieve all investigations. Examples of storage under EM can be viewed here: EM examples (pdf file).

Light microscopy

Lymphocytes from patients with JNCL caused by mutations in CLN3 contain vacuolations that can be readily seen by light microscopy of a stained peripheral blood film, particularly in the trails. To date no other NCL has been found to have vacuolated lymphocytes. Therefore if NCL has already been diagnosed then the presence of vacuolations suggests JNCL. Vacuolations are however found in lymphocytes from patients with some other lysosomal storage disorders, and care must also be taken not to confuse vacuolations with swollen mitochondria. Rectal biopsy containing nerve cells (not obtained if suction biopsy is used), as cryostat sections of frozen samples, was formerly used to determine if neuronal storage was present and to define which cell types were involved, using an acid phophatase reaction, staining with Sudan black and examination. Autofluorescence could also be detected, but care had to be taken to distinguish between normal age pigment and the NCL-specific pigment, which could be done with a good combination of filters. Light microscopic findings must be corroborated by electron microscopy and/or mutation analysis and/or enzyme analysis.

Cell lines

Establishing a cell line from a fresh skin biopsy placed in tissue culture medium is a simple procedure. An alternative approach is by transformation to make lymphoblast cell line from a blood sample. In terms of future diagnostic requirements this avoids the need for repeated samples to be taken from the patient. A cell line allows an unending supply of material that can be used to make DNA or RNA for future mutation analysis as new genes are identified, and provides cells suitable for biological research (for cell biology and microscopic studies). Establishing a cell line is recommended for cases of unusual disease course or when it has not been possible to determine the exact type of NCL, or when a rare mutation has been found.

Prenatal diagnosis

As with most genetic diseases, it is possible to carry out diagnostic tests prenatally to establish whether a baby is affected or is healthy (and whether a carrier or not) in a family that has already had an affected child or when both parents are known to be carriers of a mutation in the same NCL gene. There are three main ways that this can be done, each requiring access to chorionic villus cells (CVS - chorionic villus sampling) from the placenta: 1) Mutation analysis in families in whom the genetic defect has been identified; 2) Enzyme analysis in families already shown to have an enzyme deficiency; 3) Identification of typical inclusions by electron microscopy in families in whom there is no enzyme deficiency and the gene has not been identified. The procedure is usually performed between 10-12 weeks of a pregnancy and results may take several weeks. There is a low risk of miscarriage. If the genetic basis within a family is known, it is also possible to offer pre-implantation genetic diagnosis (PGD) combined with in-vitro fertilisation (IVF) to identify embryos that will not be affected by NCL disease.