Laboratory Issues

Due to unusual complexity in FMR1, molecular testing should be performed by an experienced molecular pathologist. If the etiology of MR in an individual is unknown, DNA analysis for FXS should be performed as part of a comprehensive genetic evaluation that includes routine cytogenetic analysis. Cytogenetic abnormalities have been identified as frequently as or more frequently than FMR1 mutations in individuals with MR who are referred for FXS testing. In addition, the use of Southern blotting on DNA isolated from amniocytes for prenatal FMR1 analysis, with typical 2- to 3-week turnaround times, may lead to stressful situations on occasion regarding the timing of possible pregnancy termination. Utilization of CVS provides additional time for possible pregnancy termination, but equivocal results sometimes occur due to incomplete methylation when a full mutation is present.

PCR-based commercial kits used to estimate repeat copy number are available through some suppliers but not widely utilized. Many laboratories use laboratory-developed methods for both FMR1 Southern blot analysis and PCR. Patient control cell lines may be purchased from the Coriell Institute (http://coriell.umdnj.edu/). Testing for FXS is routinely included in proficiency tests administered by the College of American Pathologists (CAP).

UNIPARENTAL DISOMY Molecular Basis

Several developmental disorders arise not just from classical gene mutations but also from the effects brought to bear on gene expression by chromosomal aneuploidy. Aneu-ploidy occurs in a substantial percentage of all recognized pregnancies, yet most instances result in embryonic lethality with spontaneous abortion during early pregnancy. This high rate of observed aneuploidy suggests the existence of numerous abnormal gametes, either nullisomic or disomic for a particular chromosome, due to meiotic nondisjunc-tion events. Considering the relatively high frequency of aneuploidies, Eric Engel in 1980 hypothesized the potential for rare "gametic complementation" between a gamete nullisomic for a particular chromosome and a gamete disomic for the same chromosome.7 Although derived from two separate "mistakes," such a union would lead to an apparently normal (2n or disomic) individual with inheritance of two copies of a chromosome pair (or a chromosomal segment) from one parent and no copy from the other parent, or uniparental disomy (UPD). Maternal UPD occurs when a child has two copies of one of the mother's chro mosomes and no copies of that particular chromosome from the father. Paternal UPD occurs when a child inherits two copies of a specific chromosome from the father and no copies of that chromosome from the mother.

Uniparental disomy may or may not cause developmental problems, depending on which chromosome is involved. However, patients identified with UPD indicate that the possible clinical consequences include (1) expression of recessive disorders when only one parent carries a recessive trait, (2) disorders related to parent-of-origin effects (imprinted genes), and (3) residential effects of chromosome aneu-ploidy (mosaicism). The inheritance of two identical chromosomes, or isodisomy, may occur due to meiosis II nondisjunction, formation of isochromosomes through centromeric misdivision, or mitotic nondisjunction in a monosomic diploid cell. Isodisomy is of particular concern due to the potential expression of recessive disorders when one parent is a carrier of a recessive trait, and for imprinting disorders. Inheritance of two homologous, but non-identical, chromosomes from one parent is termed heterodisomy, and occurs as a result of a meiosis I nondis-junction. The presence of heterodisomy raises concern related to expression of imprinting disorders.

Gametic complementation is one of several possible mechanisms producing UPD (Table 6-2). The most common mechanism leading to UPD appears to be trisomy rescue. Observations of mosaicism for normal and tri-somic karyotypes confined to extraembryonic (placental) tissue obtained by CVS, or confined placental mosaicism (CPM), led to recognition of trisomy rescue. Upon later cytogenetic examination of fetal or neonatal tissue, this mosaicism is not detected and has resolved into an apparently normal disomy. A trisomy may be "rescued" by loss of one trisomic set member through nondisjunction, anaphase lag, or chromosome degradation mediated by centromeric loss. Because the chromosome loss is random, the incidence of UPD in a diploid fetus with known CPM is theoretically 1 in 3. Correction or rescue of a monosomic cell line may occur through early mitotic nondisjunction or endoreduplication of a whole chromosome in a mono-somic conception. In addition, chromosomal transloca-

Table 6-2. Mechanisms Leading to UPD Trisomy rescue Monosomy rescue Gametic complementation Chromosomal translocation

• Centric fusions of acrocentric chromosomes

• Familial heterologous Robertsonian translocation

• Familial homologous Robertsonian translocation

• Heterologous de novo centric fusions

• Homologous de novo centric fusions

• Reciprocal balanced translocations De novo somatic recombination

Pericentric and paracentric inversions in imprinted domains Small marker chromosomes containing imprinted genes

Source: Reference 8.

tions, somatic recombination, inversions in imprinted domains, and marker chromosomes also may lead to UPD.

When Engel first conceptualized UPD, he calculated that perhaps 3 in 10,000 individuals have UPD for one of the chromosomes (15, 16, 21, 22, or the sex chromosomes) commonly observed in aneuploidy.7 Immediately recognized was the potential consequence of isodisomy, resulting in duplication of recessive alleles from a single carrier parent. In 1988, the discovery of cystic fibrosis (CF) in a young girl with maternal UPD for chromosome 7 was the first report of UPD resulting in a recessive condition.9 The girl's mother was a CF carrier but her father was not. Recessive conditions caused by UPD have been reported for UPD involving chromosomes 1,2,4,5,6,7,8,9,11,13,14,15,16, and X (Table 6-3).

The later discovery of genomic imprinting revealed additional pathological consequences related to UPD.10 While most genes are expressed from functional alleles derived from both parents, a small minority of genes are normally expressed only from one allele, either the maternal or paternal allele. This differential gene expression depending on the parent of origin results from imprinting, a process initiated in germinal tissue and maintained in somatic tissue by methylation of DNA. Imprinting may be tissue specific and is a normal process for regulating dosage of gene products when normal, biparental inheritance occurs. As a result of DNA methylation, control elements regulate expression of specific individual genes, or whole segments of chromosomes containing several

Table 6-3. Recessive Disorders Associated with UPD

Parent of

Disorder

Chromosome

Origin

Junctional epidermolysis bullosa

1

M, P

Chédiak-Higashi syndrome

1

M

Pycnodysostosis

1

P

Congenital pain insensitivity

1

P

with anhidrosis

5-alpha-reductase deficiency

2

P

Abetalipoproteinemia

4

M

Spinal muscular atrophy

5

P

Methylmalonic acidemia

6

P

21-hydroxylase deficiency

6

P

Complement deficiency

6

P

Cystic fibrosis

7

M

Osteogenesis imperfecta

7

M

Congenital chloride diarrhea

7

P

Lipoprotein lipase deficiency

8

P

Leigh syndrome

9

M

Cartilage hair hypoplasia

9

M

ß-thalassemia

11

M

Retinoblastoma

13

P

Rod monochromacy

14

M

Bloom syndrome

15

M

a-thalassemia

16

P

Familial Mediterranean fever

16

P

Hemophilia

X

P

Duchenne muscular dystrophy

X

M

Sources: References 8, 10.

M, maternal;P, paternal.

Table 6-4. Disorders Associated with Imprinted Genes

Maternal or

Disorder/Phenotype Gene(s) Locus Paternal UPD

Transient neonatal IGF2R 6q25-q27 Paternal diabetes mellitus Russell-Silver syndrome Beckwith-Wiedemann syndrome Maternal UPD14 syndrome (precocious puberty/ short stature) Paternal UPD14 syndrome (abnormal thorax, short stature) Prader-Willi syndrome

Angelman syndrome Intrauterine growth retardation

Sources: References 8,10.

genes, exclusively from either the maternal or paternal alleles. Uniparental disomy for chromosomes containing imprinted genes results in functional loss of gene expression even when no change to the DNA sequence has occurred. Although a small number of genes are affected, several disorders result from imprinting defects or loss of gene expression related to UPD of a chromosome containing imprinted genes (Table 6-4).

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