Molecular Testing Methods and Laboratory Issues

For the disorders associated with a limited number of mutations, testing for the point mutations can be performed by a number of methods such as restriction enzyme digestion of polymerase chain reaction (PCR) products, allele-specific PCR, allele-specific hybridization by dot-blot, or in a homogeneous assay format (e.g., TaqMan or Lightcycler). Analysis of most, but not all, of the FGFR gene

Table 9-1. A Small Selection of FGFR3 Mutations That Can Be Detected by Restriction Enzyme Digestion of the Appropriate PCR Products

Disorder

Mutation

Sequence Change

Restriction Sites

Create New

Destroy Existing

Thanatophoric dysplasia 1

R248C

CGC ^ TGC

Bsp 1286I

Hae II

Thanatophoric dysplasia 1

S371C

AGT ^ TGT

Cvi KI

Tsp RI

Achondroplasia

G380R

GGG ^ AGG

Sfc I

Bsc GI

Achondroplasia

G380R

GGG ^ CGG

Msp I

Bsc GI

Thanatophoric dysplasia 2

K650E

AAG ^ GAG

Bsm AI

Bbs I

The use of at least 2 different restriction enzymes is

advisable to ensure that all true digestion results can

be distinguished from enzyme

failure. Restriction enzyme analysis of patient DNA with the K650E mutation is shown

in Figure 9-1.

mutations can be accomplished by restriction enzyme digestion of PCR products followed by electrophoretic separation of the digestion products in agarose gels. Whenever possible, two different restriction enzymes are used, one for the normal sequence and one for the mutation sequence. This combination will always give fully interpretable banding patterns, if it can be achieved. If the above plusminus system cannot be achieved, the next best option is to design the PCR such that the PCR product contains a second constant restriction enzyme recognition site so that the product always is cleaved but yields different patterns in individuals positive or negative for the mutation. Digestion with a second restriction enzyme can be used to confirm a result; for instance, the G375C mutation in FGFR3 does not destroy a restriction enzyme site but can be detected by the generation of new sites for Sph I or Fat I. Table 9-1 gives an example of selected mutations found in FGFR3 and options for testing by restriction enzyme digestion of the DNA (refer to Online Mendelian Inheritance in Man (OMIM),2 GeneReviews,3 or Human Gene Mutation Database4 for current and complete information on mutations in the FGFR genes). Figure 9-1 shows the results of restriction enzyme analysis of DNA from normal controls

Figure 9-1. Polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) assay for the K650E mutation in exon 15 of the FGFR3 gene. Exon 15 of FGFR3 from patient and control DNA was amplified by PCR,and the resulting PCR products were either not digested or digested with Bsm Al or Bbs I, and analyzed by agarose gel electrophoresis. The K650E mutation in exon 15 of FGFR3 creates a new restriction site for Bsm Al and destroys a restriction site for Bbs I. Lane 1, size standards (50 base pair [bp] ladder); lane 2, undigested PCR product control (480 bp); lane 3, no template PCR control; lanes 4 and 6, normal DNA digested with Bsm Al; lane 5, patient DNA digested with Bsm Al, which demonstrates an additional cut site compared to the normal DNA in adjacent lanes; lanes 7 and 9, normal DNA digested with Bbs I; lane 8, patient DNA digested with Bbs I, which demonstrates loss of a cut site compared to the normal DNA in adjacent lanes. These results confirm a diagnosis of thanatophoric dysplasia, type 2.

Figure 9-1. Polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) assay for the K650E mutation in exon 15 of the FGFR3 gene. Exon 15 of FGFR3 from patient and control DNA was amplified by PCR,and the resulting PCR products were either not digested or digested with Bsm Al or Bbs I, and analyzed by agarose gel electrophoresis. The K650E mutation in exon 15 of FGFR3 creates a new restriction site for Bsm Al and destroys a restriction site for Bbs I. Lane 1, size standards (50 base pair [bp] ladder); lane 2, undigested PCR product control (480 bp); lane 3, no template PCR control; lanes 4 and 6, normal DNA digested with Bsm Al; lane 5, patient DNA digested with Bsm Al, which demonstrates an additional cut site compared to the normal DNA in adjacent lanes; lanes 7 and 9, normal DNA digested with Bbs I; lane 8, patient DNA digested with Bbs I, which demonstrates loss of a cut site compared to the normal DNA in adjacent lanes. These results confirm a diagnosis of thanatophoric dysplasia, type 2.

and a patient with the K650E mutation in FGFR3 (thanatophoric dysplasia, type 2).

The major problem with most restriction enzyme5 assays for the FGFR-related conditions is that there are often many different mutations (sometimes also in different FGFR genes) that can cause the same phenotype, so that many different assays may need to be performed until a positive result is obtained. A better approach, whenever the mutations are clustered,is to sequence the relevant exon(s). This approach will detect any mutation, including mutations not previously identified, in the region sequenced. Figure 9-2 is an automated DNA sequence that shows a C278F mutation in exon 3a of the FGFR2 gene from a patient with Crouzon syndrome. Note that this sequencing approach would have detected any of the 10 or more mutations in this exon that cause Crouzon syndrome.

A faster screening method has recently become available with the advent of denaturing high-performance liquid chromatography (DHPLC), designed for the analysis of DNA fragments. Figure 9-3 shows superimposed DHPLC traces of exon 3a of the FGFR2 gene in a normal control and patients with Apert (P253R and S252W) and Crouzon (W290R) syndromes. The P253R, S252W, and W290R mutations have traces that are clearly different from one another as well as from the control. The speed of analysis using DHPLC is a major advantage (4 minutes per sample). Although each patient has a different profile and therefore

T A G A 125

GTTTGTCTN 130 135

C A A G G T T 140

T A 145

(JL h-)s ■

à a

1500

1550 1600

1650 1700

Figure 9-2. Sequence analysis of exon 3a of the FGFR2gene.Automated sequence elec-tropherogram (from an ABI377 sequencing instrument; Applied Biosystems, Foster City, CA) of the forward strand of exon 3a of FGFR2 from a patient with Crouzon syndrome. A TGC to TTC mutation is shown at position 136 in this sequence that would cause a C278F amino acid substitution. This particular mutation creates a new restriction site for Bbs I so could also be detected by a PCR-RFLP method for diagnosis or confirmation.

Figure 9-2. Sequence analysis of exon 3a of the FGFR2gene.Automated sequence elec-tropherogram (from an ABI377 sequencing instrument; Applied Biosystems, Foster City, CA) of the forward strand of exon 3a of FGFR2 from a patient with Crouzon syndrome. A TGC to TTC mutation is shown at position 136 in this sequence that would cause a C278F amino acid substitution. This particular mutation creates a new restriction site for Bbs I so could also be detected by a PCR-RFLP method for diagnosis or confirmation.

Consolidated analysis report

mV

10

9-

7-

6 5 4 3

1_=_

;_;_min

Figure 9-3. DHPLC analysis for patients with Apert or Crouzon syndromes. Superimposed DHPLC traces of exon 3a of the FGFR2 gene in a normal control (brown trace) and patients with Apert (P253R, pink trace; S252W, green trace) and Crouzon (W290R, blue trace) syndromes. The 3 mutations, each differing from the normal control by only a single nucleotide, have different DHPLC profiles.

Figure 9-3. DHPLC analysis for patients with Apert or Crouzon syndromes. Superimposed DHPLC traces of exon 3a of the FGFR2 gene in a normal control (brown trace) and patients with Apert (P253R, pink trace; S252W, green trace) and Crouzon (W290R, blue trace) syndromes. The 3 mutations, each differing from the normal control by only a single nucleotide, have different DHPLC profiles.

a different sequence compared to the normal control, the exact mutation is not identified by DHPLC. Previously run affected controls provide a good indication of which mutation corresponds to which DHPLC profile, particularly where there is a very limited number of mutations associated with the diagnosed disorder. DNA from patients with profiles that do not match any of the reference set must be sequenced to determine the specific mutation that is identified by DHPLC.

The laboratory should ensure that the relevant family members have received adequate genetic counseling and have provided informed consent prior to testing. Regardless of the methods used for detection of FGFR mutations, in vitro diagnostic test kits are not commercially available for any of the genes or mutations. Proficiency testing programs are not currently available for FGFR testing, so the laboratory must meet proficiency testing requirements by other mechanisms.

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