Diagnosis

This website deals with clinical management, diagnosis, therapeutic options of the disease and genetic counselling in general terms. The information in this website can by no means substitute for medical advice to the individual patient or family.

A brochure which describes criteria for NBS diagnosis, intended for paediatricians and general practitioners, is available, as a downloadable and printable *.pdf file, at the end of this page. Please, after reading this page, just click on the icon to get your copy.

For a certain diagnosis of NBS besides the recognition of the characteristic phenotype, laboratory investigations are essential.

Mean age at diagnosis is around 7 years and cancer appears prior to the diagnosis in 20-30% of patients. This is a very dangerous situation since, due to their increased radiosensitivity, NBS patients are at risk of developing severe toxic complications (even death) and/or second malignancy when treated with conventional radio- and chemotherapy. This implies that:

Microcephaly. When severe microcephaly (OFC below the 3^rd percentile) of unknown origin is present at birth or develops during the first months of life, NBS diagnosis must be taken into account. In NBS microcephaly is accompanied by a peculiar facial appearance, with sloping forehead, receding mandible and prominent midface; these dysmorphisms become more obvious with age. Other associated features can help in the differential diagnosis of NBS from other forms of microcephaly.

When microcephaly is associated with
frontal lobe hypoplasia corpus callosum posterior part agenesis	major structural malformations of the brain
normal neuromotor development in the first year of life	severe psychomotor delay
	severe epilepsy with onset in early infancy
NBS	other forms of microcephaly

Please note that even if major structural malformations of the brain, severe psychomotor delay and severe epilepsy with onset in early infancy seem not to be associated with NBS and suggest a different diagnosis, their presence is not a sufficient criterion to exclude NBS diagnosis.

When NBS is suspected the use of ionising radiation for diagnostic purposes (e.g. CT scan of the brain), if not strictly necessary, must be avoided.

Recurrent respiratory infections. Recurrent respiratory infections may be the most evident symptom of NBS, preceding laboratory evidence of immune deficiency and being present at diagnosis in 30-40% of patients.

The evaluation of the immunological profile in NBS should include:

- classes and subclasses of immunoglobulins

- B and T lymphocyte subpopulations (CD19+, CD20+, CD3+, CD4+, CD8+, CD4+/CD8+, CD4+CD45RA+/CD4+CD45RO+, CD16+, CD56+)

- in vitro proliferative response of T-lymphocytes to mitogens/antigens (e.g. PHA, anti-CD3, anti-TCR)

The most common defects in NBS are: combined IgG and IgA deficiency with normal levels of IgM, low levels of IgG2 and IgG4, reduced number of CD3+ T lymphocytes with low CD4+ cells and decreased CD4+/CD8+ ratio, increased number of NK cells and greatly reduced in vitro proliferative response of T-lymphocytes to mitogens. It must be remembered that the deterioration of the immune system is often progressive in NBS and that very young patients can have completely normal immunological parameters. Hence, when NBS is suspected a monitoring of immune function is necessary.

Lymphoproliferative disorders. A lymphoproliferative disorder, mainly B- cell non-Hodgkin lymphomas (B-NHL) and acute lymphoblastic leukaemia (ALL), may be the presenting finding.

The diagnosis of NBS should be carefully considered before radio- chemotherapy is initiated in patients with lymphoproliferative disorders:

- of very young age (younger than 3 years)

- who also have congenital or developmental defects

The presence of a chromosome instability disorder, including NBS, must be suspected in any patient who develops severe adverse reactions to radio- chemotherapy.

When NBS is suspected on the basis of the clinical phenotype, cytogenetic analysis allows the detection of both spontaneous and radiation-induced chromosome instability and a confirmation of the diagnosis.
For spontaneous chromosome instability analysis, slides are obtained from peripheral blood lymphocyte cultures. At least 100 metaphases should be analysed for both chromosome and chromatid breaks (Giemsa-staining) and rearrangements (QFQ-banding). The frequency of metaphases with aberrations and the total number of breaks and rearrangements can be compared with aberration frequency observed in age-matched controls. In order to improve the quality of the analysis each laboratory should have its own controls.
In some cases, chromosome analysis may be hampered by a reduced response of T lymphocytes to mitogens.
On both lymphoblastoid cell lines and fibroblast cultures radiosensitivity can be assessed by scoring the number of induced chromatid-type aberrations (on 100 Giemsa-stained metaphases) after G2-phase treatment with low doses of radiation. The frequency of induced chromatid breaks in NBS cells is higher than in control cell lines and clearly differentiates them from healthy cells.

Western blotting assay with polyclonal antibody direct against nibrin can confirm the diagnosis of NBS, by demonstrating the lack of expression of p95 in lymphoblastoid cell lines (LCLs).
In NBS LCLs co-immunoprecipitation assay with polyclonal Nbs1 antibody allows the detection of C-terminal abbreviated Nbs1 polypeptide which maintain their ability to interact with hMre11. C-terminal proteins with different molecular weight are synthesised by different mutated alleles of the NBS1 gene, thus their detection can help to estimate the localization of the mutation/s within the NBS1 gene.

Recently, the missense mutation R215W (643C>T) has been found at the compound heterozygous state with the classic 657del5 mutation in two monozygous twins who, despite presenting with a clinically severe form of NBS, did not show chromosomal instability (spontaneous and induced). Full length nibrin was present in the lymphoblastoid cell lines of the patients, but with reduced expression. See also Disease Description (Clinical Phenotype, Cytogenetics and Cellular Phenotype, Molecular Biology).

Molecular testing enables definitive confirmation of the diagnosis, with the demonstration of disease-causing mutations in both alleles of the NBS1 gene. All disease-causing mutations identified to date are located within exons 6-10 and all but one of them result in premature truncation of the nibrin protein. Patients of Slavic origin are likely to be homozygous for the 657del5 mutation, while nine different mutations have been found in patients of diverse ethnic groups, two of them are Italian. No genotype-clinical phenotype correlation have been established to date.

Contact us if you need help with NBS diagnosis and please download your copy of the NBS diagnosis brochure for paediatricians and general practitioners by clicking on the icon below (*.pdf file).