DNA BASED MUTATION ANALYSIS FOR X

DNA BASED MUTATION ANALYSIS FOR X-LINKED ADRENOLEUKODYSTOPHY (X-ALD)

The DNA Diagnostic Laboratory at the Johns Hopkins Medical Institutions and the Peroxisome Disease Diagnostic Laboratory at the Kennedy Krieger Institute have established a collaborative program of DNA-based mutation analysis for patients with X-ALD and at-risk family members. More than 400 different mutations of the X-ALD gene have been identified and in most instances the mutation is unique in each pedigree. This, together with the occurrence of paralogues on autosomes has complicated the identification of mutations. The Johns Hopkins DNA Diagnostic Laboratory has developed a strategy to resolve these technical problems. The mutation analysis will prove valuable in prenatal testing. It is of particular value for the identification of women at risk of being carriers, since it minimizes the risk of false negative results that may occur with present biochemical diagnostic techniques.

The DNA analysis is performed on 10 ml of venous blood collected in an EDTA tube and should be mailed to the Peroxisome Disease Diagnostic Laboratory at Kennedy Krieger Institute. The first step in the process is to examine a blood sample from either a male with X-ALD (the ideal scenario) or an obligate carrier family member. With this sample a screening analysis will be performed to attempt to identify the mutation responsible for X-ALD. The cost for the screening test is specified on the requisition form and results are generally available within four to eight weeks. On rare occasions it may not be possible to identify the mutation.

Once a family’s mutation has been identified, a targeted analysis, looking specifically for that family’s mutation, can be performed for relatives. The turn around time is approximately three to four weeks using a 10 ml blood sample in an EDTA tube.

Funding for the tests must be received prior to analysis.

For further information, contact Ann Moser or Steven Steinberg at 443 923 2788.

PEX Gene Mutation Identification in Zellweger Spectrum Patients

Requisition Form for DNA Testing

Zellweger syndrome (ZS), neonatal adrenoleukodystrophy (NALD) and infantile Refsum’s disease (IRD) are the most commonly diagnosed peroxisomal biogenesis disorders (PBD). At least 12 different PEX genes are associated with this Zellweger spectrum of PBD.

We are now offering DNA sequence analysis for exons from the following PEX genes: PEX1, PEX2, PEX10, PEX12 and PEX26.

This testing is offered as a two-tiered test. First PEX1 exons 13 & 15 are sequenced. If no pathogenic mutations are detected in these exons, then we can reflex analysis to our panel of exons from PEX2 (exon 4), PEX10 (exons 4 & 5), PEX12 (exons 2 & 3) and PEX26 (exons 2 & 3).

We reported our experience using this approach to mutation identification in 91 Zellweger spectrum patients in 2004 (Steinberg et al, Molecular Genetics & Metabolism 2004, 83: 252-263). The two-tier test being offered for clinical testing has an estimated sensitivity of 70% for the identification of at least one pathogenic PEX gene mutation. The sensitivity for identifying both pathogenic PEX gene mutations is about 35-40%.

There are several potential benefits to this test.

(1) Rapid identification of the PEX gene causing the PBD. Previously this was possible only on a research basis by complementation analysis. Complementation analysis is methodologically complex, time consuming, utilizes extensive resources, and difficult to perform in a routine manner. Our sequencing approach will identify the defective PEX gene in about 70% of patients.

(2) Carrier testing for family members would be possible. Carriers for PBD do not have measurable biochemical defects. Thus, carrier testing for family members has not been possible. Relatives concerned about their carrier status can be offered DNA carrier testing if PEX gene pathogenic mutations are identified.

(3) Some genotypes may be prognostically useful. Early evidence suggests that one common PEX1 gene mutation (G843D) is associated with the milder clinical phenotypes NALD or IRD (Poll-The et al American Journal of Medical Genetics 2004, 126A:333-338). Thus, this testing may be of some prognostic value.

(4) Prenatal testing using direct CVS. The prenatal testing currently available in our laboratory requires cultured CVS or amniocytes. DNA testing could be performed using genomic DNA isolated from direct villi, thus providing the possibility of an earlier prenatal result. However, in order for prenatal testing to be performed by DNA analysis, both pathogenic mutations must be identified in the proband and confirmed in the parents.

PEX7 Gene Testing for Rhizomelic Chondrodysplasia Punctata

Requisition Form for DNA Testing

PEX7 encodes the receptor for a subset of peroxisomal matrix proteins. Without a functional PEX7, these enzymes are mislocalized to the cytosol. Mutations in PEX7 are associated with the following autosomal recessive disorders:

(1) Rhizomelic Chondrodysplasia Punctata type 1 (RCDP1)

-Clinical: proximal shortening of humerus, & to less degree femur (rhizomelia); punctate calcification of cartilage; coronal clefts of vertebral bodies; cataracts, at birth or within few months of life; severe mental retardation; postnatal growth retardation; early lethality (1-2 years); milder variants exist
-Biochemical diagnosis: deficient erythrocyte membrane plasmalogens; deficient plasmalogen synthesis in fibroblasts; deficient phytanic acid oxidation in fibroblasts; older patients, elevated plasma phytanic acid; VLCFA are normal!

(2) Adult Refsum disease (ARD)

-ARD is mainly associated with mutations in the gene that encodes phytanoyl-CoA 2-hydroxylase; only a small proportion of cases are associated with PEX7 mutations!!
-Clinical: retinitis pigmentosa; peripheral neuropathy; cerebellar ataxia
-Biochemical diagnosis: elevated phytanic acid; deficient phytanic acid oxidation

Sensitivity of PEX7 mutation analysis:

RCDP1 patients: Two common alleles are associated with exon 9: (1) L292X, 67% of patients; (2) IVS9+1G>A, 15% of patients. Two less common alleles are reported for exon 7: (1) G217R, 6.8% of patients; (2) A218V, 9.8% of patients. (Braverman et al, 2002; Motley et al, 2002). The predicted sensitivity for identifying at least a single allele in e9 alone is 85%. The predicted sensitivity for identifying at least a single allele in e7 & e9 is 90%. The sensitivity for identifying two deleterious alleles by combined e7 & e9 analysis is ~57%. The highest sensitivity is predicted for patients of Northern European descent. (Braverman et al, 2002) Less than 5% of patients with an RCDP clinical phenotype and deficient plasmalogen synthesis may have a single enzyme defect in this pathway (RCDP2 & 3). However, careful biochemical characterization can identify these patients in advance.

ARD patients: None of the 3 ARD patients reported in the literature had PEX7 mutations in exons 7 or 9 (Jansen et al, 2004).

References:
1. N. Braverman, Li Chen, Paul Lin, Cassandra Obie, Gary Steel, Pamela Douglas, Pranesh K. Chakraborty, Joe T.R. Clarke, Avihu Boneh, Ann Moser, Hugo Moser, David Valle Mutation Analysis Of Pex7 In 60 Probands With Rhizomelic Chondrodysplasia Punctata And Functional Correlations Of Genotype With Phenotype. Human Mutation 2002, 20:284-97.

2. Motley AM, Brites P, Gerez L, Hogenhout E, Haasjes J, Benne R, Tabak HF, Wanders RJ, Waterham HR. Mutational spectrum in the PEX7 gene and functional analysis of mutant alleles in 78 patients with rhizomelic chondrodysplasia punctata type 1. Am J Hum Genet. 2002, 70:612-24.

3. Braverman N, Moser A, Steinberg S (updated February 2004) Rhizomelic Chondrodysplasia Punctata Type 1 in: GeneReviews at GeneTests: Medical Genetics Information Resource [database online]. Copyright, University of Washington, Seattle. 1997-2004. Available at www.genetests.org.

4. Jansen GA, Waterham HR, Wanders RJA Molecular basis of Refsum disease: sequence variations in phytanoyl-CoA hydroxylase (PHYH) and the PTS2 receptor (PEX7). Human Mutation 2004, 23:209-218.

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