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X Linked Disease in Females J Med Genet 1993; 30: 177-184 177 ORIGINAL ARTICLES J Med Genet: first published as 10.1136/jmg.30.3.177 on 1 March 1993. Downloaded from X chromosome inactivation and the diagnosis of X linked disease in females R M Brown, G K Brown Abstract larly important in the brain which has an In studies of female patients with sus- obligatory requirement for aerobic glucose ox- pected deficiency of the Elm subunit of idation under normal circumstances. Rela- the pyruvate dehydrogenase complex, we tively modest reduction in PDH activity there- have found that X inactivation ratios of fore has significant consequences, especially 80:20 or greater occur at sufficient fre- for central nervous system function. quency in cultured fibroblasts to make Within the PDH complex, the Ela subunit exclusion of the diagnosis impossible in contains the pyruvate binding site and the about 25% of cases. Pyruvate dehydroge- phosphorylation sites by which the activity of nase Elm subunit deficiency is an X linked the whole complex is controlled.5 The gene for inborn error of metabolism which is well the Elca subunit is located on the short arm of defined biochemically and is unusual in the X chromosome in the region Xp22. 1.6 that most heterozygous females manifest However, in all reported series of patients with the condition. The diagnosis is usually PDH Ela deficiency, there are approximately established by measurement of enzyme equal numbers of males and females.2- The activity and the level of immunoreactive clinical presentation does differ between the protein and these analyses are most com- sexes with the acute metabolic form more monly performed on cultured fibroblasts common from the patients. Skewed patterns of X in males and neurological presenta- chromosome inactivation make it impos- tions predominating in females. The disorder sible to exclude the diagnosis if the nor- in females, although generally less severe, still usually results in death before adulthood and mal X chromosome is expressed in the http://jmg.bmj.com/ majority of cells. While most of the only a few subjects with mutations in the Ela observed variation appears to be the gene are known to have had children. PDH expected consequence of random X inac- Ela deficiency therefore represents an unusual tivation, it may be further exaggerated case of an X linked disease involving a house- by sampling and subsequent expansion of keeping gene product, in which almost all the cells for analysis. cases are sporadic and where the threshold (J7 Med Genet 1993;30:177-84) for developing symptoms is so low that it is usually exceeded in heterozygous females. on September 30, 2021 by guest. Protected copyright. The diagnosis of PDH Ela deficiency is There are relatively few X linked diseases usually established in the first instance by which are expressed in most heterozygous measurement of enzyme activity in samples females and for which the responsible gene and from the patients. Most commonly, cultured its product have been defined. As a conse- fibroblasts from a skin biopsy are used, al- quence, little attention relatively has been results obtained with given to problems which might arise in this though lymphoid cells or tissue are situation when diagnosis is based on measure- biopsy samples occasionally reported. In with ment of the function or structure of the gene conjunction these enzyme product. One example of an X linked disease assays, most laboratories also determine the which does behave in this way is pyruvate levels of immunoreactive protein correspond- dehydrogenase (PDH) Elca subunit deficiency. ing to the different subunits of the complex. This is a relatively common inborn error of Subsequently, the underlying mutation may metabolism with a broad spectrum of clinical be defined but, at present, direct analysis ofthe basic Genetics Laboratory, presentation ranging from severe lactic aci- gene defect is not feasible as a primary Department of dosis in the newborn to a progressive neuro- screening procedure. Biochemistry, degenerative disease with prolonged survival.l1 For reliable diagnosis of PDH Ela defi- University of Oxford, South Parks Road, The PDH complex is an important and ciency in females, it is essential that each X Oxford OX1 3QU. widely distributed housekeeping enzyme chromosome is represented in the active form R M Brown which catalyses the conversion of pyruvate to in a significant proportion of the cells in the G K Brown acetyl coenzyme A within the mitochondrion. sample. Increasing deviation away from ap- Correspondence to This is a key regulatory step in the central proximately equal representation towards a Dr G K Brown. pathways of energy production in the cell and situation in which the normal X chromosome Received 15 July 1992. Revised version accepted the enzyme operates at close to its maximal is expressed in the majority of cells will make it 17 August 1992. activity in a number of tissues. This is particu- progressively more difficult to identify the 178 R M Brown, G K Brown presence of an El x mutation against the back- ment of the buffer concentration, or samples ground of considerable normal variation in were digested first with PstI and then ethanol enzyme activity. In practice, less than 20% precipitated and resuspended in Boehringer representation of an X chromosome with a null buffer L for the second digestion with MspI or J Med Genet: first published as 10.1136/jmg.30.3.177 on 1 March 1993. Downloaded from EhIo mutation would not be detectable. HpaII. Samples were separated by running on In this paper, we compare methods for X 0 5% agarose for 42 to 46 hours using 1 25 V/ inactivation analysis in cultured human fibro- cm. blasts and assess the significance of the For analysis of X inactivation patterns using observed variation in the pattern of X inactiva- the PGK probe, a double digest with PstI and tion for the diagnosis of PDH Elax deficiency BstXI was performed, followed by digestion in females based on experience with 48 with HpaII'°; 12 pg samples were digested at patients. We also present data on X chromo- 37°C overnight (- 15 hours) with PstI. BstXI some inactivation patterns in chorionic villus enzyme was added and the second incubation cell cultures which indicate that these are often was carried out for eight hours at 55°C. Sam- unsuitable for the diagnosis of X linked disease ples were then ethanol precipitated and resus- based on measurements of gene expression. pended in Boehringer buffer L and divided into two aliquots. One aliquot was subse- quently digested with HpaII and the other was Materials and methods left undigested. Samples were separated by CELL CULTURE running in 1 1% agarose for - 22 hours at Fibroblasts from forearm skin biopsies were 1 5 V/cm. cultured in Basal Medium (Eagle) with 10% Initial densitometric measurements of rela- fetal calf serum, 100 units/ml penicillin, and tive band intensity indicated that the X inacti- 100 pg/ml streptomycin. Cultures were always vation ratio could be reliably determined to split at high density to minimise the possibility within 10%. Comparison of densitometric of expanding small cell populations and intro- tracings with ratios estimated by eye indicated ducing a sampling bias. The cultures were that visual classification was equally accurate regularly screened for mycoplasma contamina- as a semiquantitative measure and this was tion using the method of Chen.7 Chorionic subsequently used to group ratios into centile villus cell cultures were established in Chang bands. At least two different times of exposure medium from cleanly dissected villi. They of the x ray film were used in estimating the were subsequently cultured in Ham's F10 ratios and in many cases several different ana- medium containing 20% fetal calf serum, 2% lyses of the patient samples were performed Ultraser G (Gibco), 100 units/ml penicillin, with the various methods. and 100 ptg/ml streptomycin. The possibility of clonal expansion during subsequent subcul- ture was again reduced by ensuring that the PROBES cultures were never allowed to grow up from The M27P probe used to detect the DXS255 http://jmg.bmj.com/ low density. locus was a 2 3 kb EcoRI insert prepared from the subclone M27f3." The PGK probe, pSPT/ PGK, was an 800 bp BamHI/EcoRI fragment ENZYME ASSAY from the 5' end of the PGK gene'0 and was Overall PDH activity was measured after max- kindly supplied by Dr B Vogelstein. imal activation with dichloroacetate, using [1- as substrate as described '4C]-pyruvate pre- on September 30, 2021 by guest. Protected copyright. viously.8 In this assay, the activity of normal Results control fibroblasts ranges from 0 7 to 1 1 nmol METHYLATION PATTERN ANALYSIS 14CO2 produced/mg protein/min. All samples were analysed for methylation at the DXS255 locus using the M271 probe. Double digests were performed with MspI or X INACTIVATION ANALYSIS HpaII and either BamHI or PstI. The DNA was prepared from fibroblast cell pellets DXS255 locus is contained within a 30 to using a standard proteinase K-SDS method. 35 kb BamHI fragment and within this MspI/ Restriction enzymes were purchased from HpaII sites immediately flanking the VNTR Boehringer Mannheim and digestion was car- region of the locus are separated by 6 to 12 kb. ried out using a 3 to 5 fold excess of enzyme in Methylated DNA which is not digested by the manufacturer's recommended buffer sys- HpaII remains in the high molecular weight tem. For analysis of X inactivation patterns (30 to 35 kb) band and different alleles are not using the M27,B probe, MspI/BamHI and usually resolved. Unmethylated DNA (on in- HpaII/BamHI double digests were performed active X chromosomes) is cut to generate a two as described previously.9 Briefly, the MspI and band pattern (figs 1 to 3A).'2 The methylation HpaII digests were carried out overnight.
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