Diagnosis of ALD

April 7th, 2016 |

Hugo W Moser, M.D. Ann B Moser, B.A. Steven J Steinberg, Ph.D. and Stephan Kemp Ph.D.

Clinical diagnosis

The diagnosis of ALD should be considered in four distinct clinical settings:

Neuroimaging

Brain MRI is always abnormal in neurologically symptomatic males and often provides the first diagnostic lead. In approximately 85% of affected individuals, MRI shows a characteristic pattern of symmetrical enhanced T-2 signal in the parieto-occipital region with contrast enhancement at the advancing margin.

Control MRIMRI of an X-ALD patient with severe cerebral involvement

Very long-chain fatty acids

Males: The most important laboratory assay is the measurement of the concentration of very long-chain fatty acids (VLCFA) in plasma. VLCFA levels are elevated in 99.9% of males with ALD of all ages regardless of the presence or absence of clinical symptoms. The three parameters analyzed are: the concentration of C26:0, the ratio of C24:0/C22:0, and the ratio of C26:0/C22:0. Table 1 shows the mean results for normal controls, affected males, and carrier females [Valianpour et al. 2003].
Analysis of VLCFA is extremely specialized and therefore it is performed only in a few laboratories worldwide.

Normal Males with ALD Women with ALD
C26:0 µmol/L 0.67 +/- 0.13 2.94 +/- 0.87 1.54 +/- 0.72
C24:0/C22:0 ratio 0.86 +/- 0.13 1.52 +/- 0.21 1.18 +/- 0.15
C26:0/C22:0 ratio 0.01 +/- 0.003 0.05 +/- 0.02 0.02 +/- 0.01
Table 1: VLCFA concentrations determined in controls and ALD patients using electrospray ionization mass spectrometry (ESI-MS) (Valianpour et al. 2003).

Plasma VLCFA levels

Important comment from Dr. Ann Moser: Lorenzo’s oil, a mixture of erucic and oleic acids, is still used in some countries to normalize VLCFA levels. The Peroxisome Disease laboratory at the Kennedy Krieger Institute in Baltimore routinely reports erucic acid (C22:1) levels when measuring plasma VLCFA. Certain oils used in cooking, such as mustard seed oil, have naturally high levels of erucic acid and, thus, can lead to an elevation similar to that observed during Lorenzo oil therapy.

Women with ALD: Increased concentration of VLCFA in plasma and/or cultured skin fibroblasts is present in approximately 85% of women with ALD; 15% of known women with ALD have normal plasma concentration of VLCFA. The average plasma VLCFA results obtained from women with ALD are shown in Table 1. The discriminant function reported in Moser AB et al (1999) is not able to distinguish all women with ALD from the normal control range (see the Figure). Women should be tested genetically when ALD is suspected and VLCFA concentrations are normal.

Mutation analysis

The ABCD1 gene is the only gene associated with ALD. More than 700 different mutations have been identified in ABCD1 [Kemp et al. 2001]. Many ALD kindred’s have a unique mutation. All proven pathogenic mutations identified in the ABCD1 gene are catalogued on this website.

Boehm and colleagues have developed and validated a robust DNA diagnostic test for ALD involving non-nested genomic amplification of the ALD gene, followed by fluorescent dye-primer sequencing and analysis. The method covers all coding exons and the flanking intron-exon junctions in 10 separate amplicons [Boehm et al. 1999]. This protocol provides a highly reliable means of determining carrier status in women at risk for transmitting ALD and is applicable to a clinical diagnostic laboratory. This method has become the diagnostic sequence-based analysis of choice for many laboratories worldwide.

Genetic counseling

ALD is inherited in an X-linked manner. The ABCD1 gene is the only gene associated with ALD.
Parents of a male or female patient: About 93% of index cases have inherited the ABCD1 mutation from one parent; at most, 7% of individuals with ALD have de novo mutations. It is appropriate to measure plasma VLCFA levels in the mothers of both affected males and affected females and in the fathers of affected females (Figure below). When the disease-causing mutation has been identified in an affected family member, mutation analysis of the ABCD1 gene can be used in the evaluation of the parents and for extended family screening.
Sibs of an index patient (proband): The risk to sibs depends upon the genetic status of the parents, which can be clarified by pedigree analysis, VLCFA measurement, and molecular genetic testing.
If the proband’s mother is a carrier, the chance of transmitting the disease-causing mutation in each pregnancy is 50%. Male and female sibs who inherit the mutation will be affected.
If the proband’s father has a disease-causing mutation in the ABCD1 gene, all of the female sibs will be affected and none of the male sibs will be affected.
If neither parent is a carrier, the risk to sibs of a proband is low.
Offspring of a proband: Affected males transmit the ABCD1 mutation to all of their daughters and none of their sons.
Affected females have a 50% chance of transmitting the ABCD1 mutation in each pregnancy. Sons who inherit the mutation will be affected; daughters who inherit the mutation are carriers and will most likely develop symptoms in adulthood.

Figure 2: (Left) If a woman is a carrier for ALD she has the following possible outcomes with each newborn: when the child is a daughter, there is a 50% chance that the daughter is a carrier for ALD and a 50% chance that the daughter is unaffected. In case the child is a boy, there is a 50% chance that the son has ALD and a 50% chance that he will be unaffected. (Right) For an X-linked disorder, such as ALD, if an affected man has children, then all of his sons will not have the disease (he always passes his Y-chromosome on to his son). But all of his daughters will be carriers (he always passes his only (affected) X-chromosome on to his daughter).

Left image: If a woman is a carrier for ALD she has the following possible outcomes with each newborn: when the child is a daughter, there is a 50% chance that the daughter is a carrier for ALD and a 50% chance that the daughter is unaffected. In case the child is a boy, there is a 50% chance that the son has ALD and a 50% chance that he will be unaffected. Right image: For an X-linked disorder, such as ALD, if an affected man has children, then all of his sons will not have the disease (he always passes his Y-chromosome on to his son). But all of his daughters will be carriers (he always passes his only (affected) X-chromosome on to his daughter).

Carrier identification

Testing of at-risk female relatives for carrier status is a two-step process. Measurement of plasma concentration of VLCFA is performed first; if abnormal, the female is a affected. Because around 15% of women with ALD have normal plasma concentration of VLCFA, molecular genetic testing should be used to test those females with a normal concentration. This is easier if the disease-causing ABCD1 mutation has already been identified in the family.

Extended family testing

Depending upon their gender, family relationship, and the carrier status of the proband’s parents, the proband’s aunts and uncles and their offspring may be at risk of being carriers or of being affected.

Evaluation of at-risk family members is important for management and genetic counseling but is often implemented insufficiently. Several factors may contribute to insufficient evaluation:

Prenatal testing

Prenatal diagnosis to detect a possible affected male fetus can be offered to women whose carrier status has been clearly confirmed by genetic analysis of the ABCD1 gene. In some countries, noninvasive prenatal determination of fetal sex using cell-free fetal DNA present in maternal blood may be performed at 7 weeks of pregnancy. It relies upon the detection of Y chromosome sequences by PCR techniques. If the fetus is a male, ABCD1 mutational analysis can be performed on a fresh chorionic villus sample (CVS) at 11–13 weeks of pregnancy. Alternatively, the determination of fetal sex can be performed by conventional cytogenetic techniques on a CVS sample at 11–13 weeks of pregnancy and then ABCD1 mutational analysis performed if the fetus is a male. Mutation analysis takes a few days. Prenatal diagnosis can also be done on amniotic cells at 15–18 weeks of gestation. This approach, however, requires at least an additional 2–3 weeks of amniotic cell culture in order to generate enough cell material for molecular analysis. In case the ABCD1 mutation has not yet been identified in the family, but the carrier status of the female has been clearly established biochemically by the demonstration of increased VLCFA levels in her plasma, prenatal diagnosis of a male fetus has to be done by the measurement of VLCFA levels in cultured CVS cells or amniotic cells. Preimplantation genetic diagnosis can also be offered in some countries for selected cases. Usually this is done when an affected female has had at least two prenatal diagnoses leading to pregnancy interruption because the fetus was an ALD male. A significant number of women with ALD develop a myelopathy in adulthood and prenatal diagnosis of a female fetus to establish whether the fetus has ALD may be offered on an individual basis. This situation occurs more and more frequently when either the father or the pregnant mother has severe clinical symptoms.

Newborn screening

A newborn screening method has been developed. It can detect elevated VLCFA (as C26:0-lysoPC) in bloodspots. In 2014, New York State started newborn screening for ALD. In 2015, the Netherlands expanded its newborn screening program from 17 to 31 conditions, including ALD. On February 16, 2016, ALD was added to the United States Recommended Uniform Screening Panel (RUSP). Early diagnosis of ALD is the key to saving lives, because newborn screening allows prospective monitoring and early intervention.

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