Am. J. Hum. Genet. 48:335-341, 1991

X-linked Sideroblastic Anemia and Ataxia: Linkage toPhosphoglycerate Kinase at Xq 13Wendy H. Raskind, * Ellen Wijsman, t Roberta A. Pagon,t't Timothy C. Cox, 1 Michael J. Bawden,Brian K. May, 1 and Thomas D. Birdt'§

Department of Medicine, Divisions of *General Internal Medicine, tMedical Genetics, and $Pediatrics, University of Washington School of Medicine,and §Division of Neurology, Veteran's Administration Hospital, Seattle; and IlDepartment of Biochemistry, University of Adelaide, Adelaide

Summary

Molecular linkage analysis was performed on a kindred with X-linked sideroblastic anemia and ataxia.Two-point analysis with a DNA probe for phosphoglycerate kinase (PGK1), which maps to Xql3, suggestedlinkage to the disorder by a lod score of at least 2.60 at a recombination fraction of zero. The disease in thiskindred appears to be clinically and genetically distinct from that in previously reported families withX-linked hereditary ataxia or spastic paraparesis. No mapping data are available for inherited X-linkedsideroblastic anemia without neurologic abnormalities. However, structural alterations of band Xql3 maybe involved in the development of idiopathic acquired sideroblastic anemia. No alterations in the restrictionpatterns of two X-linked genes involved in erythrocyte formation-i.e., a DNA-binding protein (GF-1) and5-aminolevulinate synthase (ALAS) -were detected in DNA from affected males, arguing against a largedeletion in either of these candidate genes.

Introduction

The sideroblastic anemias are a heterogeneous groupof conditions characterized by a microcytic, hypo-chromic anemia and abnormal accumulation of ironin mitochondria of bone marrow erythroblasts (Bot-tomley 1982). Sideroblastic anemias may be acquiredor inherited in an X-linked recessive or, very rarely,autosomal manner (Kasturi et al. 1982). The heredi-tary spastic parapareses and ataxias can be autosomalor X linked and can be either pure or complicated byother abnormalities (Harding 1983, 1984). We pre-viously described an X-linked recessive sideroblasticanemia with nonprogressive ataxia (XLSA/A; Pagonet al. 1985). In the present paper we present the find-ings of molecular linkage analysis in family 1 of Pagonet al. (1985), with probes detecting X chromosome-

Received April 17, 1990; final revision received September 17,1990.

Address for correspondence and reprints: Wendy H. Raskind,M.D., Ph.D., Department of Medicine, RG-20, University ofWashington, Seattle, WA 98195.i 1991 by The American Society of Human Genetics. All rights reserved.0002-9297/91 /4802-0018$02.00

specific RFLPs. Preliminary experiments with twocandidate genes for sideroblastic anemia were alsoperformed.

Methods

Kindred

The clinical presentation has been reported else-where (see family 1 in Pagon et al. 1985). Affectedmales have infantile or early childhood onset of non-progressive marked cerebellar ataxia and mild spas-ticity without sensory loss or mental retardation.Prominent cerebellar atrophy is shown in the MRIscan of family member III-1 (fig. 1). The anemia ismild, with hypochromia and microcytosis associatedwith raised free erythrocyte protoporphyrin levels butlack of excessive parenchymal iron storage in adult-hood. A pedigree updated to include additional familymembers is shown in figure 2. Patient IV-12 was bornfollowing the original report of the family. At 9 mo ofage anemia was diagnosed. Red cell indices were asfollows: hemoglobin, 10.7 g/dL; hematocrit, 34%;mean corpuscular volume, 63 .t3; mean corpuscularhemoglobin, 20 pg; and mean corpuscular hemo-

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Raskind et al.

Figure I Magnetic resonance image of family member 111-1at 41 years of age. Marked atrophy of the superior cerebellar vermisis demonstrated, with normal brain stem, cerebral cortex, and uppercervical spinal cord. Additional views (not shown) revealed normalappearing white matter.

globin concentration, 32%. Values of iron studieswere as follows: total iron, 48 mg/dL (normal 55-

155 mg/dL); total iron binding capacity, 290 mg/dL; and iron saturation, 16.6% (normal 20%-50%).Protoporphyrin-to-heme ratio was greater than 250.Ferritin was 26 ng/ml. At 10 mo of age truncal ataxiawas apparent when the patient was sitting and stand-ing. Five motor development appeared to be normal.XLSA/A syndrome was diagnosed.

DNA Analyses

Mononuclear and polymorphonuclear (PMN) leu-kocytes were separated from EDTA-anticoagulated

II

1 2 324 6 7 10 1 12 13 14154 16 17IV 0S OCIS00 0 2 1040

Figure 2 Pedigree of kindred with XLSA/A.0 = Affectedmale; ( = obligate carrier female; O! = ring sideroblasts in marrow;9 = normal marrow exam; * = DNA obtained; § = neonataldeath; t = Smith-Laemli-Optiz syndrome; : = died of pneumoniaat 18 mo-clinical status unknown.

blood by centrifugation on a discontinuous Histo-paque (Sigma, St. Louis) gradient (specific gravities.1.077 and 1.119). B-lymphoblastoid cell lines wereestablished (Raskind et al. 1984), and DNA was ex-tracted from them and from uncultured PMNs (Peru-cho et al. 1981). In one case, individual IV-4, DNAwas extracted from hair follicles. Restriction-endo-nuclease digestions were performed according to therecommendations of the enzyme supplier (BoehringerMannheim Diagnostics, Houston; New England Bio-labs, Cambridge, MA; and Pharmacia Fine Chemi-cals, Piscataway, NJ). Southern blot analyses ofrestriction-fragment alleles were performed by a modi-fication of the methods described by Vogelstein et al.(1987). Formamide was not used; to achieve the ap-propriate stringency, hybridizations and washes weredone at proportionately higher temperatures (Pui et al.1989); and blots were prepared on Zeta-Probe nylonfilters (Bio-Rad, Richmond, CA). Human DNA in-serts for the probes RB-0.8 (PGK1), RC8 (DXS9),p22-23 (DXS1 1), p58-1 (DXS14), pX65H7 (DXS72),p754 (DXS84), p87.1 (DXS164), p87.15 (DXS164),Xfx301b (DXS311), pRN1 (DXS369), and pDP34(DXYS1X) were cut from plasmids and were 32p oligo-labeled (Feinberg and Vogelstein 1983) with a reagentkit (Pharmacia Fine Chemicals) for Southern blot anal-yses of RFLPs. X-inactivation studies with a PGK1RFLP were performed according to a method de-scribed elsewhere (Pui et al. 1989).

Candidate genes GF-1 and erythroid ALAS havebeen localized to Xp21-11 and Xp2l-Xq2l, respec-tively (Zon et al. 1989; Cox et al. 1990). Southernanalyses were performed with the full-length humancDNA clones. DNAs from affected and unaffectedfamily members were digested with EcoRI, TaqI,MspI, HindIII, and PstI to search for evidence of adetectable deletion in these genes.

Linkage analysis was performed with LIPED (Ott1976) under a model of recessive, X-linked inheritanceof XLSA/A. All di. pnoses were made prior to initia-tion of the linkage studies. The gene frequency ofXLSA/A used in the analysis was .001, and pene-trance in males and homozygous females was assumedto be complete. The analyses were performed twice.A conservative analysis was done with the set of ob-served phenotypes, without consideration of bonemarrow findings. A second analysis was subsequentlydone under the assumption that two unaffected fe-males who had either no sons (III-4) or only unaffectedsons (III-3) were obligate carriers because their mar-rows contained abnormal mitochondrial iron loading

336

X-linked Sideroblastic Anemia and Ataxia

as manifested by perinuclear localization of coarse sid-erotic granules in ring sideroblasts. Such abnormalcells are not present in normal marrow (Cartwrightand Deiss 1975) but were seen in the two obligateheterozygotes (11-2 and 111-2) tested in this family.However, we do not know the frequency with whichcarriers of X-linked sideroblastic anemia manifest thislaboratory finding.

Results

The genotypes obtained with each of the informa-tive probes are shown in table 1. The X-chromosomelocation and two-point lod scores for these probes are

given in table 2. Under the conservative analysis, a

maximum lod score of 2.60 at a recombination frac-tion (0) of 0 was obtained for the marker PGK1 (thefirst probe assayed). Under the more liberal analysis inwhich all women whose marrows contained abnormalring sideroblasts were identified as carriers, the maxi-mum lod score for PGK1 reached 3.60 at 0 = 0. The

computed 1 -lod-unit support intervals (Conneally etal. 1985) were 0 = 0-.17 (lod score = 1.60) and 0= 0-. 15 (lod score = 2.55) for the conservative andliberal analyses, respectively. A maximum lod score

of 0.90 was found for the probe pX65H7, locatedat Xq21.1. However, this probe detected only threeinformative meioses. The remaining nine probes ex-

cluded close-to-moderate linkage to XLSA/A.Southern blot analysis of the candidate loci GF-1

and ALAS by using five restriction enzymes gave iden-tical patterns with digests ofDNA from affected malesand their unaffected relatives. This result shows thata gross deletion (more than 100 bp) in one of these twogenes is not responsible for XLSA/A in this family.X-chromosome inactivation analysis of a PGK1 BstXI

RFLP was performed on granulocyte DNAs from fiveobligate carrier females (11-2, 111-2, 111-5, 111-6, and111-7). There was no evidence for selection against cellswith an active maternal (affected) chromosome. Theproportion of inactive maternal alleles revealed by di-gestion with the methylation-sensitive enzyme HpaIIwas 46%-64%.

Table I

Genotypes

GENOTYPE OBTAINED WITH INFORMATIVE PROBEaPEDIGREE AND

INDIVIDUAL RC8 p87.1 p87.15 p754 p58-1 PGK1 pX65H7 pDP34 p22-33 Xfx301b pRN1

II:2. AB AB B B AB AB A A AB AB A3. B A ... B A ... ... B ... A B4. A B B B A A B A B A A

III:1. B A B B B B A A B B A2. B AB AB AB AB AB A AB B AB A3. B AB AB AB AB AB A AB B AB A4. AB B AB AB B AB A AB B AB A5. AB A B B AB AB AB AB A A AB6. AB AB B B A AB AB AB A AB AB7. AB AB B B A AB AB AB A A AB8.................B A B B A A AB AB A AB AB

IV:3. B B AB A A A AB B B A A4 .. . . .......... . . . . . . . . A . .. . ... . . . . .

5. B A B B B B A A B B A6.................B A B AB AB AB A AB B AB A7............. ... B B ... A ... A ... ...

8. ... ... B B ... A ... B ... ... ...

13. ... B AB AB A AB AB B AB A AB14.A A B B A B A A A A B15.B B B B A B A B A A A16.AB AB B AB A A B AB AB A A

a A = larger fragment; B = smaller fragment.

337

Raskind et al.

Table 2

Two-Point Linkage Tests

LOD SCORE AT 0 OF

GROUP AND MARKER (band) .00 .001 .05 .1 .2 .3 .4

A:aRC8 (p22.2) ...................... -00 - 7.30 -2.13 -1.23 -4.18 -.08 .04p87.1 (p21.2) ...................... -00 -6.92 -1.95 -1.16 -.49 -.17 -.02p87.15 (p21.2) .................... - 00 - 7.23 - 2.15 -1.28 -.50 -.14 .00p754 (p21.1) ...................... .00 .00 .02 .04 .06 .06 .04p58-1 (pll.21) .................... - 00 -7.44 - 2.37 - 1.50 -.70 - .31 - .10PGK (q13) ...................... 2.60 2.59 2.31 2.02 1.41 .80 .27pX65H7 (q21.1).................. .90 .90 .82 .73 .54 .36 .18pDP34 (q21.31) ................... - 00 - 2.44 -.70 -.39 -.09 .03 .05p22-33 (q24-q25)................. - 00 -4.70 - 1.38 -.86 -.41 -.19 -.06Xfx301b (q26-qter) ............... -cc0 -7.65 -2.53 - 1.63 -.78 -.35 -.11pRN1 (q27.2-27.3) .............. -cc0 -2.44 -.75 -.47 -.21 -.08 -.02

B: bRC8 (p22.2) ...................... -c - 9.90 - 3.03 - 1.83 -.73 -.23 -.02p87.1 (p21.2) ...................... -00 -9.32 -2.67 - 1.61 -.68 -.25 -.04p87.15 (p21.2) .................... - 00 - 9.23 - 2.59 - 1.53 - .62 - .21 - .26p754 (p21.1) ...................... 00 -5.30 - 1.88 - 1.26 -.66 -.32 -.12p58-1 (pll.21) .................... -00 - 9.84 - 3.09 -1.95 -.90 -.38 -.11PGK (q13) ...................... 3.60 3.59 3.27 2.92 2.17 1.35 .50pX65H7 (q21.1).................. .90 .90 .82 .73 .54 .36 .18pDP34 (q21.31)................... - -4.74 - 1.35 -.77 -.26 -.04 .04p22-33 (q24-q25)................. - 00 -7.10 -2.06 - 1.23 -.49 -.16 -.02Xfx301b (q26-qter) ............... -00 -6.65 - 1.72 -.98 -.42 -.21 -.10pRN1 (q27.2-27.3) .............. - 00 - 2.44 -.75 -.47 -.21 -.08 -.02

a Only women with affected sons are considered obligate carriers.b All women with abnormal marrow sideroblasts are considered to be carriers.

Discussion

Most hereditary ataxias and spastic parapareses are

inherited as autosomal dominant or recessive traits(Harding 1983; Chamberlain et al. 1988), but occa-

sional families with an X-linked mode of inheritancehave been reported. Even the X-linked recessive formshave shown genetic heterogeneity, differing in age at

onset, severity of disease, rate of progression, andpresence of additional abnormalities (Gutmann et al.1990). Our family differs clinically from previouslyreported kindreds, even those whose primary neuro-

logic symptom is ataxia (Shokeir 1970; Spira et al.1979; Farlow et al. 1987; Young et al. 1987). Thereis evidence from published linkage studies that muta-tions in at least two genes in the mid and terminalportions of the long arm of the X chromosome may

produce syndromes of spasticity (table 3; Kenwrick etal. 1986; Keppen et al. 1987; Goldblatt et al. 1989;

Le Merrer et al. 1989). One form of X-linked spasticparaparesis appears to be linked to DXYS1 (probepDP34) and DXS17 (probes S21 and S9) in Xq21.31-q22. Another form shows linkage to DXS52 (probeSt14) at Xq28; St14 was not informative in our family,and negative lod scores were obtained for pDP34. Wedid not evaluate probes S21 and S9. Both the pheno-type (i.e., nonprogressive ataxia, minimal spasticity,lack of mental retardation, and marked cerebellar at-rophy) in our family and the mapping data that indi-cate the involvement of a gene in a more proximalregion of the X chromosome long arm (Xql 3) suggestthat this syndrome is not an allelic variant of thosepreviously reported.

There are no mapping data on the inheritedX-linked form of sideroblastic anemia without neuro-logic involvement. Structurally rearranged X chromo-somes have been reported in myelodysplasias andacute leukemias with sideroblastic hemopoiesis. Band

338

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340 Raskind et al.

Xql3 is a recurring breakage site in these cases (Sessar-ego et al. 1983; Knapp et al. 1985; Mackinnon et al.1988; Dewald et al. 1989). Therefore, it is postulatedthat a gene in band Xq13 may be responsible for thisphenotype. Since the phenotype in the XLSA/A familyis complex, with both neurologic and hematologic ab-normalities, the genetic defect may involve an alter-ation oftwo contiguous genes, e.g., by an overlappingdeletion.We evaluated the restriction patterns of two

X-linked genes that are expressed in erythroid cells, todetermine whether a detectable deletion is present inDNA from affected males. Both genes are probablylocated on the X short arm. However, it seemed rea-sonable to consider them candidate genes, since theconfidence interval around Xq1 3 as the site for XLSAIA is wide, given the small size of the family. GF-1,assigned to Xp21-11, is a cell-specific DNA-bindingprotein believed to be involved in transcription regula-tion (Zon et al. 1989). The erythroid-specific hemebiosynthetic enzyme ALAS maps within Xp21-Xq21,most likely at Xpl1.2 (Cox et al. 1990). The conver-sion of glycine and succinyl CoA to 5-aminolevulinateby ALAS is the first step in porphyrin ring formation.It has been postulated that a defect in this enzymemay be one cause of hereditary X-linked sideroblasticanemia (Lee et al. 1968; Aoki et al. 1973; Cox et al.1990). Southern blot analysis of DNA from affectedmales did not reveal a difference in band pattern foreither ALAS or GF-1. Therefore, it is unlikely that alarge deletion in or near either of these genes is presentin this family. Nonpolymorphic as well as polymor-phic DNA sequences mapped to Xql3 are now beingused to screen for possible deletions in this region ofthe chromosome. On the other hand, since mutationof a single gene may produce pleiotropic effects indifferent cell lineages, a point mutation or microdele-tion in GF-1 or ALAS might be the molecular basisfor XLSA/A. The nucleotide sequences of the codingregions of these genes are being further examined inaffected males.

AcknowledgmentsWe appreciate the excellent technical assistance of Gabriel

Herner, Hillary Lipe, Mark Matsushita, Catherine Morgan,and John Wolff. This research was supported by grant CA16448 from the National Institutes of Health, by VeteransAdministration Medical Research Funds, and by grantsfrom the American Health Assistance Foundation and fromthe National Health and Medical Research Council of Aus-

tralia. Probes were provided by Drs. K. Fischbeck, L. Kun-kel, S. Orkin, D, Page, P. Pearson, B. Schmeckpeper, J.Singer-Sam, R. Sood, and J. Zon. We are especially gratefulto the affected family, whose willing cooperation made thisstudy possible.

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