Available Assays

Mutation of the NF1 gene is the only known cause of the disorder. Molecular tests for diagnostic, prenatal, and preimplantation diagnosis are available. The choice of assay and testing laboratory depends upon the reason for referral and mutation types and detection rates of their assay(s).

Fluorescent in situ hybridization (FISH; see chapter 2) with NF1 probes of either metaphase or interphase white blood cells is the optimal test to rule out or confirm the approximately 5% of cases due to a submicroscopic NF1 microdeletion (Figure 21-1).18 In the future, a first-tier test may employ an NF1 deletion junction-specific polymerase chain reaction (PCR) assay.19 The recent availability of high-resolution genomic microarrays of the NF1 deletion region will facilitate clinical testing by array-comparative genomic hybridization (CGH),20 which may become clinically important in the future if deletions involving a subset of genes predispose to certain manifestations. The sensitivity of deletion-specific PCR and array-CGH assays to detect low-level NF1 deletion mosaicism will need to be determined. Routine cytogenetic analysis is of limited clinical utility, as the NF1 microdeletions are submicroscopic, and translocation and rearrangement involving NF1 are extremely rare.

Linkage analysis is an indirect test that tracks the inheritance of the mutant NF1 allele in members of a family. This may be the quickest, most economical NF1 test for at-risk individuals and fetuses of families that fulfill the testing criteria. The primary requirement is the availability and cooperation of multiple family members whose NF1 status is known by detailed clinical evaluation. Multiple NF1 intragenic polymorphic markers are available that facilitate identification and tracking of the predisposing haplo-type in a family and provide the specificity for linkage testing.

Efficient detection of subtle intragenic NF1 gene mutations, for purposes of diagnostic testing or mutation typing for prenatal or preimplantation diagnosis, is complicated by the large number of exons and large size of the gene (Table 21-1), variation in type and distribution of mutations, and large fraction of private mutations. About 70% to 80% of mutations result in a premature translation termination codon, with nonsense and splicing defects being the most common.21 These mutations can be detected by the protein truncation test (PTT; see chapter 2), which detects truncated neurofibromin polypeptides synthesized by in vitro translation of multiple overlapping NF1 complementary DNA (cDNA) segments. A detection rate of about 80% can be attained with an optimized PTT testing protocol (see Laboratory Issues below). The majority of such mutations are private to each individual or family, although there are recurrent mutations that may account for, at most, a few percent of cases (Figure 21-1a).

About 10% of NF1 mutations are missense or in-frame insertions or deletions of a few nucleotides,21,22 some of which show clustering (Figure 21-1a). Their identification requires direct sequence analysis of NF1 exons and splice junctions in genomic DNA or cDNA segments. Prospective testing of NF1 subjects by direct genomic sequence analysis revealed a detection rate of 89%, which is more streamlined than PTT testing and allows for automation.23 Various mutation scanning techniques of NF1 genomic DNA or cDNA are employed by clinical laboratories, including denaturing high-performance liquid chromatography (DHPLC), temperature gradient gel electrophoresis (TGGE), single-strand conformation polymorphism (SSCP), and heteroduplex analysis (HA) (see chapter 2). Although high detection rates are reported in the literature using DHPLC (72-95%),24,25 it is important to realize that the detection rates for mutation scanning protocols will be laboratory specific due to the degree of optimization of the specific technique. A survey of clinical laboratories is recommended prior to sample submission. DHLPC has the advantages of using genomic DNA and high-throughput capability compared to the cDNA/gel-based PTT; however, a recently reported high-throughput PTT may be available for clinical testing in the future.26

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