Molecular Vision 2006; 12:159-76 ©2006 Molecular Vision Received 13 October 2005 | Accepted 8 March 2006 | Published 10 March 2006

CHST6 mutations in North American subjects with macular corneal dystrophy: a comprehensive molecular genetic review

Gordon K. Klintworth,1,2 Clayton F. Smith,2 Brandy L. Bowling2

Departments of 1Pathology and 2Ophthalmology, Duke University Medical Center, Durham, NC

Purpose: To evaluate mutations in the carbohydrate sulfotransferase-6 (CHST6) gene in American subjects with macular corneal dystrophy (MCD). Methods: We analyzed CHST6 in 57 patients from 31 families with MCD from the United States, 57 carriers (parents or children), and 27 unaffected blood relatives of affected subjects. We compared the observed nucleotide sequences with those found by numerous investigators in other populations with MCD and in controls. Results: In 24 families, the corneal disorder could be explained by mutations in the coding region of CHST6 or in the region upstream of this gene in both the maternal and paternal chromosome. In most instances of MCD a homozygous or heterozygous missense mutation in exon 3 of CHST6 was found. Six cases resulted from a deletion upstream of CHST6. Conclusions: Nucleotide changes within the coding region of CHST6 are predicted to alter the encoded protein signifi- cantly within evolutionary conserved parts of the encoded sulfotransferase. Our findings support the hypothesis that CHST6 mutations are cardinal to the pathogenesis of MCD. Moreover, the observation that some cases of MCD cannot be explained by mutations in CHST6 suggests that MCD may result from other subtle changes in CHST6 or from genetic heterogeneity.

Macular corneal dystrophy (MCD; OMIM 217800), a polysaccharidoses [18], and analyses of sulfated GAGs pro- rare inherited disorder first described in 1890 by Groenouw duced by organ cultures of corneas with MCD led to the dis- [1], has been identified throughout the world. MCD is most covery that corneal tissue with MCD does not synthesize KS prevalent in India [2-4] and Saudi Arabia [5]. In some coun- [19] or normal KS containing proteoglycans (PGs) because of tries, this disease accounts for a high percentage (10-75%) of defective sulfation [20,21]. Immunochemical studies using an the corneal dystrophies requiring keratoplasty [6,7]. Clinically, antibody that recognizes antigenic KS (AgKS) disclosed het- MCD is characterized by a cloudiness of the cornea and ir- erogeneity among cases of MCD based on the reactivity of regularly shaped superficial opacities of both eyes that pro- corneal tissue with the antibody [22]. Subsequently this led to gressively extend through the entire thickness of the central the recognition of three immunophenotypes that are clinically and peripheral corneal stroma. The corneal stroma is thinner and histopathologically indistinguishable from each other than normal [8-10]. In 1938, its autosomal recessive mode of [5,22-24]. Most often neither the serum nor the corneal tissue inheritance became appreciated [11]. More than two decades contain AgKS (MCD type I), but sometimes AgKS is absent later Jones and Zimmerman [12,13] differentiated MCD his- in the corneal stroma and the serum but can be detected in the topathologically from the other two major stromal corneal keratocytes (MCD type IA). A third immunophenotype (MCD dystrophies known as granular and lattice corneal dystrophy. type II) is characterized by the presence of AgKS in corneal Histopathologically, MCD is typified by an intracellular stor- tissue and detectable serum levels of AgKS that are often age of glycosaminoglycans (GAGs) within keratocytes and present in normal amounts. MCD type I corneas have been the corneal endothelium combined with an extracellular depo- found to synthesize an abnormal KS-PG with sition of similar material in the corneal stroma and Descemet’s lactosaminoglycan side chains that lack sulfate. In sharp con- membrane [5,14]. Guttae are common on Descemet’s mem- trast, an MCD type II cornea produced a normal ratio of KS- brane [15]. In 1964, because of similarities to the systemic PG to dermatan sulfate-PG, but the net synthesis of PGs was mucopolysaccharidoses, Klintworth and Vogel [16] suggested below normal [25]. However, the chondroitin/dermatan sul- MCD might be a mucopolysaccharidosis localized to the cor- fate side chains on decorin were sulfated. Subsequently, fluo- nea. A metabolic defect in keratan sulfate (KS), the major cor- rochrome-assisted carbohydrate electrophoresis disclosed that neal GAG [17], was suspected because of the histochemical the KS chain size within the cornea and cartilage in MCD attributes of the corneal accumulations and that the cornea type I was reduced and chain sulfation was absent [26]. In a was the only site of overt abnormalities. Cell culture studies cornea with MCD type II, the sulfation of N-acetylglucosamine disclosed differences from the systemic muco- and galactose was significantly reduced and the chain size was also reduced, but to a lesser degree than in MCD type I [26]. Correspondence to: Gordon K. Klintworth, MD, PhD, Duke Univer- The absence or paucity of sulfate in KS and KS-contain- sity Medical Center, Box 3802, Durham, NC, 27710; Phone: (919) ing PGs in corneas with MCD suggested that patients with 684-3550; FAX: (919) 684-9225; email: [email protected] MCD were deficient in a carbohydrate sulfotransferase that 159 Molecular Vision 2006; 12:159-76 ©2006 Molecular Vision catalyzes the transfer of sulfate groups from 3'- Because the gene responsible for MCD was suspected of phosphoadenosine 5'-phosphosulfate (PAPS) to KS in a man- encoding for a carbohydrate sulfotransferase and had been fine ner comparable to which sulfate esters are put on other en- mapped to the same region as CHST5 and CHST6, these genes dogenous and exogenous substrates (PAPS + R-OH ->PAP + became logical suspects for the MCD disease gene. In 2000,

R-OSO3). An analysis of the serum in patients with MCD type Akama et al. [33] discovered mutations in CHST6 in MCD I disclosed normal levels of enzymatic activity for sulfating at and also found insertional or deletional defects in the region least one of the two sugars present in KS and an enzyme defi- between CHST5 and CHST6 in some cases. Subsequently, cient for sulfating N-acetylglucosamine (GlcNAc) was thought several laboratories confirmed these observations [2-4,39-52]. to be present [27]. Because the serum KS is considered to be This report documents an analysis of CHST6 in 57 pa- derived from cartilage, the discovery of undetectable levels of tients from 31 families from the United States with MCD, and AgKS in the serum of individuals with MCD type I suggested reviews these findings with all previously documented muta- that the KS sulfotransferase deficiency was not restricted to tions. the cornea. Direct evidence of cartilage involvement was ob- tained later in cartilage from the nose and ear in MCD type I METHODS [26,28]. Cartilage lacks abnormal accumulations comparable Subjects: We analyzed CHST6 in 141 individuals (57 affected to those in the cornea, but the chondrocytes and extracellular patients, 57 carriers [parents or children], and 27 normal blood matrix do not contain AgKS and the KS content of cartilage is related family members) from 31 families with MCD. In all at least 800 times lower than normal [28]. instances, the diagnosis of MCD was made on a combination Using families from Iceland, where MCD was the most of the characteristic clinical features together with the typical frequent indication for penetrating keratoplasty [7], the gene histopathologic findings in the corneal tissue obtained follow- for MCD type I was mapped to chromosome 16 (16q22) and ing a penetrating keratoplasty in one or both eyes. All patients linkage data hinted that MCD type II was also at this locus were born in the United states and some ancestral lines could [29]. Fine mapping refined the location of the MCD gene. be traced to England, Ireland, Germany, Holland, Northern Two candidate genes (TAT and LCAT) in that part of chromo- Ireland, Scotland, Switzerland, and Norway. Nonmolecular some 16 were excluded [30-32]. genetic studies have been previously reported on some of these A sulfotransferase suspected of being defective in MCD cases [14,16,18,19,53-56]. This study was approved by the would belong to the galactose/N-acetylgalactosamine/N- Internal Review Board of Duke University and conformed to acetylglucosamine 6-O-sulfotransferase family of carbohy- the tenets of the declaration of Helsinki. Written informed drate sulfotransferases. This family of enzymes catalyzes 6- consent was obtained from all participants. To evaluate the O-sulfation on the 6-hydroxyl of GlcNAc, galactose (GAL), CHST6 gene in 11 deceased or unavailable patients from five or N-acetylgalactosamine (GalNAc) [33]. All known families with MCD, we analyzed the DNA of surviving sib- sulfotransferases contain highly conserved regions [34], in- lings, parents, and children to establish which mutations had cluding a 5'-PSB (phosphosulfate-binding) and a 3'-PB (3'- been transmitted to the carriers. We also analyzed DNA from phosphate binding domain) [35]. The carbohydrate 50 control specimens from the United States and DNA from sulfotransferases in different species possess marked sequence 10 normal spouses who married into these families. similarities at the amino acid level particularly in their cata- Determination of MCD immunophenotypes: Prior to mo- lytic domains and, with the exception of CHST3 (also known lecular genetic studies, the immunophenotypes had been de- as GST-0), the coding sequences of the open reading frames termined in 32 of the 57 subjects with MCD by ascertaining (ORFs) for all CHST genes are within a single exon [36]. The the presence or absence of sulfated epitopes in KS in the cor- galactose/N-acetylgalactosamine/N-acetylglucosamine 6-O- neal tissue and/or serum. The reactivity of formalin-fixed par- sulfotransferases have been designated as belonging to the GST affin-embedded corneal tissue sections with the 5D4 anti-KS family [36], but this term is not favored because they can be monoclonal antibody was evaluated immunohistochemically confused with glutathione transferases. at a dilution of 1:5,000 using the avidin-biotin One potential candidate for the MCD disease gene was immunoperoxidase complex method [22]. For negative con- CHST1 (also known as GST-1) that encodes for KS 6-O- trols, diluted normal mouse serum was used. Serum AgKS sulfotransferase, but it could be excluded because its gene had levels were measured in 22 patients with MCD with an ELISA been mapped to human chromosome 11 (11p11.1-11.2) [37]. using an anti-KS monoclonal antibody (5D4; ICN Biomedi- Further research on carbohydrate sulfotransferases resulted in cal Inc., Costa Mesa, CA) directed against a highly sulfated finding a cluster of three carbohydrate sulfotransferases with epitope present on long KS chains [57]. These immunochemi- apparent tissue restrictions on human chromosome 16 [33]. cal studies were assessed in a masked fashion without clinical CHST4, which encodes high endothelial cell GlcNAc 6-O- information or knowledge of the serum levels of AgKS. sulfotransferase, was mapped to human chromosome 16 DNA isolation: The coding region and upstream region (16q23.1-23.2) [36]. Shortly thereafter, two more highly ho- of CHST6 in 57 patients, 57 carriers (parents or children), and mologous carbohydrate sulfotransferases genes were identi- 27 unaffected blood relatives from 31 families with MCD were fied in the same region by independent investigators [33,36]. screened for mutations. In 46 patients with MCD, CHST6 was These genes were CHST5 (also known as GST-4α and intesti- analyzed on their own DNA, and it was done indirectly in 11 nal GST) [38] and CHST6 (also designated GST-4β) [36,39]. other affected subjects by studying the DNA of carriers re- 160 Molecular Vision 2006; 12:159-76 ©2006 Molecular Vision lated to them. Ten spouses of patients or their family mem- samples of genomic DNA were amplified using a protocol for bers were also screened. DNA was isolated from blood rela- the whole genome amplification (WGA) kit GenomiPhi (GE tives of 11 patients who were no longer available for study. Healthcare, Piscataway, NJ, formerly Amersham Biosciences) DNA was extracted from peripheral blood leukocytes using [58-61]. the Puregene™ Blood Kit (Gentra Systems, Minneapolis, MN) Molecular analysis of the coding region of CHST6: The or from buccal samples using the Puregene™ Buccal Cell Kit coding region of CHST6 was amplified in three overlapping (Gentra Systems) as previously described by Liu et al. [40]. fragments from genomic DNA by the polymerase chain reac- DNA was quantitated using the NanoDrop® ND-1000 UV- tion (PCR) using the same three pairs of primers as Akama et Vis Spectrophotometer (NanoDrop Technologies, Wilmington, al. [33] (primers 17 and 19, 18 and 21, 20 and 22; Figure 1, DE). Table 1). Each PCR was carried out in a 50 µl reaction mix- Analyses with insufficient DNA: In cases when insuffi- ture using the components of the Qiagen Taq DNA Polymerase cient DNA was available for a complete analysis of CHST6, Kit (Qiagen, Valencia, CA). Each reaction consisted of 1X

TABLE 1. SEQUENCES OF PRIMERS USED IN ANALYSIS OF CHST6

Primer Sequence (5'-3') Description Remarks ------1 R: TGCTGAATGGCTAACTGAAGGAATACTATAC Region A 5'-end R1M [33] 2' F: GGCCAAGTTCAGGTCAGCTTCCA Region A 3'-end F2M [33] 2 F: CCACAGCCAATTCCATCTTGGATTTTCTC Region A 3'-end F2M replacement Present paper 3 F: CCACAGAAGGAAGGACAGAGTAAATGAA Region B 5'-end F1 [33] 4 R: TTCCCTTTACTATTATAAAAATGCTGCTAATG Region B 5'-end R1 [33] 5 F: CATATCCTGTCTGGCCTAAACCTTAGTTTAC Region B 3'-end F2 [33] 6' R: GGGCACAGACAGAGGGAAAAACC Region B 3'-end R2 [33] 6 R: CATTAGACACCTCACCTGCTTTGGC Region B 3'-end R2 replacement Present paper 7 F: CAACCGGCTCCTTCCTTC Exon 1 encompassing splice sites Present paper 8 R: TCGAGACAAAAGGGGTTCTC Exon 1 encompassing splice sites Present paper 9 F: TGCACATCCCATCCTACAGA Exon 2 encompassing splice sites Present paper 10 R: CTGTGGGTTTGCAAGGAGAC Exon 2 encompassing splice sites Present paper 11 F: CTCCCCACTGAGACCATCAT Exon 3 5'-end encompassing splice site Present paper 12 R: GGTGCTGGTTGAAGAGTTGG Exon 3 5'-end encompassing splice site Present paper 13 F: CTTGAATCTGTCCCTCGTGA Exon 3 3'-end encompassing splice site Present paper 14 R: CCAGACTTGTGAGAAGAACATTTG Exon 3 3'-end encompassing splice site Present paper 15 F: TGACCCCATCTGTGGTATTG Exon 4 encompassing splice sites Present paper 16 R: CTGGTGGAAATTGGAAGCTG Exon 4 encompassing splice sites Present paper 17 F: GCCCCTAACCGCTGCGCTCTC Coding Region Part A CK71h-intrn [33] 18 F: GACGTGTTTGATGCCTATCTGCCTTG Coding Region Part B CK71h-F1041 [33] 19 R: GGCTTGCACACGGCCTCGCT Coding Region Part A CK71h-R1180 [33] 20 F: CTCCCGGGAGCAGACAGCCAA Coding Region Part C CK71h-F1355 [33] 21 R: CGGCGCGCACCAGGTCCA Coding Region Part B CK71h-R1674 [33] 22 R: CTCCCGGGCCTAGCGCCT Coding Region Part C CK71h-R1953 [33]

The numbers in the first column correspond to those in the placement diagram (Figure 1).

Figure 1. Placement of primers used in analysis of CHST6 and upstream regions. Diagram showing place- ment of primers used in analysis of CHST6 and its possible upstream rearrangements. Anomalous chro- mosomal crossovers can result in the deletion of Region B and CHST5 or the substitution of Region A for Re- gion B. These rearrangements can be detected by various combinations of forward and reverse primers 1-6. Primers 7-16 were used for analy- sis of splice sites. Primers 17-22 were used for analysis of the cod- ing region.

161 Molecular Vision 2006; 12:159-76 ©2006 Molecular Vision

PCR Buffer containing 1.5 mM MgCl2, 1X Q solution, 0.2 amplicons were sequenced to determine if any nucleotide mM dNTPs, 0.2 µM of each primer, 1 unit of Taq DNA poly- changes were present (Figure 1, Table 1). merase (Qiagen), and 200-800 ng of genomic DNA. Amplifi- Determination of chromosomal location of mutations: cation was performed in a PTC-225 PeltierThermal Cycler Two approaches were used to determine whether detected (MJ Research, Waltham, MA). PCR conditions for all three mutations were on the same or different chromosomes. When amplicons of the coding region were as follows: 2 min at 95 possible, genomic DNA from the parents, children, and sib- °C and 31 cycles of 1 min at 95 °C, 1 min at 60 °C, and 1 min lings were analyzed. Parents, children, and siblings of those at 72 °C, followed by a 4 °C hold cycle. affected with a heterozygous CHST6 mutation were all pre- The resulting amplified PCR products were purified us- sumed to be carriers of the MCD disease gene. When speci- ing QIAquick® PCR Purification Kit (Qiagen) and then se- mens of DNA were not available from appropriate family quenced on both strands using a fluorescent Big Dye Termi- members, we determined whether multiple mutations were on nator Cycle Sequencing system (Applied Biosystems, Foster the same or different chromosomes by digesting PCR prod- City, CA) combined with an ABI 377 PRISM DNA Sequenc- ucts with Surveyor nuclease (Transgenomic, Inc., Omaha, NE), ing instrument (Applied Biosystems). The sequences were then which cleaves double-stranded DNA at mismatched nucle- aligned to the CHST6 cDNA using the SeqWeb Sequence otides [63]. Amplicons containing the mutations were heat Analysis web-based program (Accelrys, San Diego, CA) to denatured and mixed with amplicons from controls. After an- seek changes in the nucleotide and amino acid sequences of nealing, the heteroduplexes were digested with Surveyor nu- exon 3 in comparison to the published cDNA sequence of clease, and the products were sized on an agarose gel. CHST6 (GenBank accession number NM_021615) and re- ported mutations. To help evaluate the significance of identi- RESULTS fied CHST6 nucleotide changes in patients with MCD, we CHST6 mutations: The most frequent abnormalities found in compared them with the sequences found in other genes en- this study were single base changes in the coding region of coding the related family of carbohydrate sulfotransferases and CHST6 that altered a coded amino acid. We identified 23 such with the protein sequences of conserved domains in the Con- examples in exon 3 of CHST6 (Table 2, Table 3). served Domain Database (CDD) within the Entrez system Single nucleotide polymorphisms: We identified four NCBI Conserved Domain Database [62]. single nucleotide polymorphisms (SNPs) that did not affect Analysis of genomic DNA sequence upstream of the cod- an amino acid (c.258A>C [Ala86Ala], c.294C>G [Ser98Ser], ing region of CHST6: Genomic DNA was screened for rear- c.465G>A [Arg155Arg], and c.681C>T [Gly227Gly]) and rangements upstream of the coding region of CHST6 accord- which were presumably insignificant as known mutations ing to the method of Akama et al. [33] using the same condi- could account for MCD in the affected individuals. In 14 indi- tions as described. The primer pairs were the same except that viduals with MCD from five families (family 7, 13, 15, 22, primer number 2' was replaced by primer number 2, and primer and 26), c.484C>G (Arg162Gly) was associated with number 6' was changed to primer number 6 (Figure 1, Table c.599T>G (Leu200Arg), but c.484C>G (Arg162Gly) was 1). HotStarTaq® DNA polymerase (QIAGEN) was used and clearly not pathogenic. Arg162Gly was found in 11 blood rela- the annealing temperatures were adjusted to 58 °C and 62 °C tives of persons with MCD and six of 50 American controls for the primer pair number 8 and number 14 and the primer were heterozygous for this nucleotide change. Moreover, an pair number 2 and number 6, respectively. In our experience unaffected 25-year-old son of a person with MCD in family the changing of primers number 2' and number 6' to number 2 26 was homozygous for this change. He had no vision diffi- and number 6 yielded more consistent results, perhaps because culties and a visual acuity of 20/15 when last examined at 23 the original primers were subject to potential mispairing due years of age. The unaffected parent was heterozygous for to the high degree of sequence similarity of CHST5 and CHST6 Arg162Gly and has no other nucleotide changes in CHST6. in this location. All PCR amplicons were electrophoresed on Another unaffected 36-year-old sister of a patient with MCD 2% agarose gels, and the gels were documented using the in family 28 was also homozygous for this change. Her 61- BioChemi Image Acquisition and Analysis Software (UVP year-old mother was heterozygous for Arg162Gly and carried BioImaging Systems, Upland, CA). All amplicons indicating Cys246Trp on the other chromosome, but had no corneal dis- upstream DNA rearrangements were sequenced to confirm ease. An unaffected daughter of a patient in family 7 was ho- their nucleotide order. Because two of the resulting amplicons mozygous for Arg162Gly and heterozygous for Leu200Arg. with our substituted primer pairs extended into less homolo- Her unaffected parent was heterozygous for Arg162Gly and gous areas, their sequences could be more readily verified. has no other nucleotide changes in CHST6. Evaluation of splice regions: In cases of MCD that could In the analysis of the splice sites, two previously reported not be explained on the basis of mutations in either the coding SNPs were detected in the 3'-UTR region of exon 4 region of CHST6 or upstream of CHST6, possible splice site (c.3342A>T, rs424964 and c.3501G>A, rs10871313), which mutations were evaluated by performing PCR. This involved presumably are common variants in the general population. using primer pairs that covered the exons and their splice sites Some unaffected family members were homozygous for these (exon 1 [primer number 7 and 8], exon 2 [number 9 and 10], SNPs. We have annotated nucleotide changes which are down- exon 3 [primers number 11 and 12, and number 13 and 14] stream from the coding region based on NM_021615 as it is and exon 4 [primers number 15 and 16]). The resulting considered the best estimate of the mRNA boundaries at this 162 Molecular Vision 2006; 12:159-76 ©2006 Molecular Vision

TABLE 2. SUMMARY OF MUTATIONS IDENTIFIED IN INDIVIDUALS AFFECTED WITH MCD IN PRESENT STUDY

Serum Family Case MCD AgKS Protein number number type (ng/ml) Alleles Nucleotide change change Zygosity ------1 1 I 1,2 c.365A>C Gln122Pro Homozygous

2 ND 1 Upstream Deletion Heterozygous 2 c.365A>C Gln122Pro Heterozygous

2 3- 4 I <9 1,2 c.392C>T Ser131Leu Homozygous

3 5 ND 1 c.271_273delGCTinsA Ala91fs Heterozygous 2 c.392C>T Ser131Leu Heterozygous

4 6- 8 I 0- <1 1 c.217G>C Ala73Pro Heterozygous 2 c.231G>A Trp77X Heterozygous

5 9 ND 1,2 Deletion Including ORF Major Homozygous

6 10-11 ND ? c.294C>G Ser98Ser Heterozygous 1 c.465G>A Arg155Arg Heterozygous 1 c.484C>G Arg162Gly Heterozygous 1 c.744C>G Ser248Arg Heterozygous 2 c.599T>G Leu200Arg Heterozygous

7 12-14 I <2- 6 1 c.1A>T Met1? Heterozygous 2 c.484C>G Arg162Gly Heterozygous 2 c.599T>G Leu200Arg Heterozygous

8 15 I 1,2 c.607G>A Asp203Asn Homozygous

9 16 ND 1 c.484C>G Arg162Gly Heterozygous 2 c.599T>G Leu200Arg Heterozygous 1,2 c.1000C>T Arg334Cys Homozygous

10 17 ND 1 c.51delG Gln18fs Heterozygous 2 c.1046G>A Cys349Tyr Heterozygous

11 18 ND 1,2 Upstream Deletion Homozygous

12 19-21 I <1- <3 1,2 c.189C>G His63Gln Homozygous 1,2 c.340C>T Arg114Cys Homozygous

13 22-23 I 1- 2 1 c.484C>G Arg162Gly Heterozygous 1 c.599T>G Leu200Arg Heterozygous 2 c.740delG Ala247fs Heterozygous

14 24 I 0 1,2 c.529C>T Arg177Cys Homozygous

15 25-26 I <2 1 c.1A>T Met1? Heterozygous 2 c.484C>G Arg162Gly Heterozygous 2 c.599T>G Leu200Arg Heterozygous

16 27-28 I ? Upstream Deletion Heterozygous ? c.1A>T Met1? Heterozygous

17 29 ND 1 c.529C>T Arg177Cys Heterozygous 2 c.815G>A Arg272His Heterozygous

18 30 ND 1,2 Deletion Including ORF Major Homozygous

19 31-32 ND 1 Upstream Deletion Heterozygous 2 c.158C>T Ser53Leu Heterozygous

20* 33 ND 1 c.137T>C Leu46Pro Heterozygous 2 c.277C>A Arg93Ser Heterozygous

21 34-35 I <3 1,2 c.1047C>G Cys349Trp Homozygous 36-37 ND 1,2 c.1047C>G Cys349Trp Homozygous 163 Molecular Vision 2006; 12:159-76 ©2006 Molecular Vision time (Personal communication, L. A. Pennacchio, DOE Joint Insertions, deletions, frameshifts, and substitutions: Genome Institute, June, 2005). Nucleotide deletions were found in the coding region of CHST6 Number of mutations in coding region: Although the cod- in family 10 (c.51delG) and family 13 (c.740delG). An inser- ing region of CHST6 in MCD most often contained a single tion was detected in family 23 (c.573_574insC), and family 3 mutation, more than one nucleotide change on a single chro- had a complex mutation with a combination of a deletion plus mosome was detected in 11 families (families 6, 7, 9, 12, 13, an insertion at the same site (c.271_273delGCTinsA). Nucle- 15, 22, 23, 26, 28, and 30). In family 20, some unaffected otide insertions within exon 3 of CHST6 (c.573_574insC) members contained two SNPs in the same chromosome, nei- caused a frameshift in family 23 (Ala192fs). Nucleotide dele- ther of which changed an amino acid. In three families (fami- tions were found in the coding region of CHST6 in four fami- lies 9, 12, and 30), the coding region of CHST6 contained two lies. Single nucleotide deletions resulted in frameshifts in fam- nucleotide changes within the same chromosome, both of ily 10 (c.51delG, Gln18fs) and family 13 (c.740delG, which were consistent with disease-producing mutations Ala247fs). The entire coding region was deleted in family 5 (Ser131Leu plus Thr228Asp [family 30], Leu200Arg plus and family 18. In family 5, exons 1 and 4 were amplifiable Arg334Cys [family 9], and His63Gln plus Arg114Cys [fam- with PCR, but in family 18, none of the 4 exons were ily 12]). amplifiable. A deletion of 3 nucleotides and an insertion of 1

TABLE 2. CONTINUED.

Serum Family Case MCD AgKS Protein number number type (ng/ml) Alleles Nucleotide change change Zygosity ------22 38-39 I <1 1 c.1A>T Met1? Heterozygous 2 c.484C>G Arg162Gly Heterozygous 2 c.599T>G Leu200Arg Heterozygous

23 40 ND ? c.258A>C Ala86Ala Heterozygous ? c.274G>C Val92Leu Heterozygous ? c.573_574insC Ala192fs Heterozygous

24 41-42 II 233-276 1,2 None None N/A

25 43 ND 1 c.91C>T Pro31Ser Heterozygous 2 c.363C>G Phe121Leu Heterozygous

26 44-45 I 0- <4 1 c.484C>G Arg162Gly Heterozygous 1 c.599T>G Leu200Arg Heterozygous 2 c.1000C>T Arg334Cys Heterozygous

27 46-47 II 282-284 1,2 ND None Unknown

28* 48 ND 1 c.484C>G Arg162Gly Heterozygous 1 c.827T>C Leu276Pro Heterozygous 2 c.738C>G Cys246Trp Heterozygous

29* 49-52 ND 1 c.1000C>T Arg334Cys Uncertain 2 Unknown Unknown Uncertain

30* 53 II 1 c.392C>T Ser131Leu Uncertain 1 c.681C>T Gly227Gly Uncertain 1 c.682A>G; 683C>A Thr228Asp Uncertain 2 Unknown Unknown Uncertain 54-56 ND 1 c.392C>T Ser131Leu Uncertain 1 c.681C>T Gly227Gly Uncertain 1 c.682A>G; 683C>A Thr228Asp Uncertain 2 Unknown Unknown Uncertain

31* 57 II 207 1 c.607G>A Asp203Asn Uncertain 2 Unknown

The nomenclature of the mutations is provided according to Antonarakis et al. [74] and the current recommendation of the Human Genome Variation Society. Accordingly, we refer to the A of the ATG-translation initiation codon of CHST6 as nucleotide +1. For amino acid number- ing, the codon for the initiator methionine is codon 1. In the table, “ND” is not determined, “N/A” is not applicable, the “?” indicates uncertain, and the asterisk indicates that mutations determined from DNA of relatives because DNA of affected individuals was not available for analy- sis. 164 Molecular Vision 2006; 12:159-76 ©2006 Molecular Vision nucleotide (c.271_273delGCTinsA) in Family 3 resulted in a tion of sequence variants), but it could not be determined frameshift (Ala91fs). A nucleotide substitution (c.231G>A) whether or not they were on separate chromosomes. A ho- in family 4 generated a heterozygous stop codon (Trp77X) mozygous upstream deletion was present in family 11. which is predicted to encode a truncated sulfotransferase. Findings in splice regions: Splice site mutations were Mutations 5'-UTR upstream of CHST6: Four families not found in the two cases of MCD from family 24 which (families 1, 11, 16, and 19) had deletions upstream of CHST6. could not be explained by mutations in the coding region of In all cases these were 40 kb deletions from a region upstream CHST6 or upstream of the gene. of CHST5 to a homologous region upstream of CHST6 (Fig- Evaluation of maternal and paternal chromosomes: Be- ure 2). In two of them, the deletions were heterozygous and cause MCD is an autosomal recessive disorder, all affected each was associated with an additional mutation in the coding individuals are expected to inherit a mutant gene from both region of CHST6 on the other chromosome (Ser53Leu, the mother and the father. In 24 of the 31 families the MCD Gln122Pro). One was found in a boy whose grandmother suf- could be explained on the basis of two or more mutations in fered from MCD due to homozygous Gln122 Pro mutation the CHST6 coding region and/or by an upstream deletion (Table (family 1). A single copy of this mutation was inherited by the 2, Table 3). Of the seven families that were not proven to have grandson, and the upstream deletion was inherited from his CHST6 mutations on both chromosomes, one family (family father. In family 16, there was a heterozygous upstream dele- 23) was found to have two mutations. We were unable to es- tion and a heterozygous c.1A>T mutation (Met1?; Human tablish whether they were or were not on the same chromo- Genome Variation Society discussions regarding the descrip- some because there was not enough DNA to amplify. In one

TABLE 3. TYPES OF MUTATIONS IDENTIFIED IN INDIVIDUALS AFFECTED WITH MCD IN PRESENT STUDY Protein Nucleotide change change Amino acid group change Mutation type ------c.1A>T Met1? Sulfur-containing - ? Missense c.91C>T Pro31Ser Aliphatic - hydroxylic Missense c.137T>C Leu46Pro Aliphatic - aliphatic Missense c.158C>T Ser53Leu Hydroxylic - aliphatic Missense c.189C>G His63Gln Basic - amidic Missense c.217G>C Ala73Pro Aliphatic - aliphatic Missense c.231G>A Trp77X N/A Nonsense c.258A>C Ala86Ala None SNP c.271_273delGCTinsA Ala91fs N/A Deletion + Insertion c.274G>C Val92Leu Aliphatic - aliphatic Missense c.277C>A Arg93Ser Basic - hydroxylic Missense c.294C>G Ser98Ser None SNP c.340C>T Arg114Cys Basic - sulfur containing Missense c.363C>G Phe121Leu Aromatic - aliphatic Missense c.365A>C Gln122Pro Amidic - aliphatic Missense c.392C>T Ser131Leu Hydroxylic - aliphatic Missense c.465G>A Arg155Arg None SNP c.484C>G Arg162Gly Basic - aliphatic SNP c.51delG Gln18fs N/A Deletion c.529C>T Arg177Cys Basic - sulfur containing Missense c.573_574insC Ala192fs N/A Insertion c.599T>G Leu200Arg Aliphatic - basic Missense c.607G>A Asp203Asn Acidic - amidic Missense c.681C>T Gly227Gly Aliphatic - aliphatic SNP c.682A>G; 683C>A Thr228Asp Hydroxylic - acidic Missense c.738C>G Cys246Trp Sulfur-containing - aromatic Missense c.740delG Ala247fs N/A Frameshift c.744C>G Ser248Arg Hydroxylic - basic Missense c.815G>A Arg272His Basic - basic Missense c.827T>C Leu276Pro Aliphatic - aliphatic Missense c.1000C>T Arg334Cys Basic - sulfur containing Missense c.1046G>A Cys349Tyr Sulfur-containing - aromatic Missense c.1047C>G Cys349Trp Sulfur-containing - aromatic Missense Deletion Including ORF Major N/A Deletion (ORF) Upstream deletion Deletion (Upstream) Amino acid group changes and mutation types for mutations listed in Table 2. In the table, “N/A” indicates not applicable. 165 Molecular Vision 2006; 12:159-76 ©2006 Molecular Vision

Figure 2. Chromosomal deletions and replacements. This diagram depicts how anomalous chromosomal crossovers in the region of the CHST5 and CHST6 genes, caused by mispairing due to homologous areas, lead to chromosomal deletions and replacements. A: Alignment of region A, CHST5, region B, and CHST6 on paired chromosomes. B: Alignment of region A with region B due to homologous areas spanning approximately 1-2 kb on either side of regions A and B (boxed area). C: Double crossover upstream and downstream from mis-paired regions A and B. D: Single crossover downstream from mis-paired regions A and B. E: Chromosome with region A replaced by region B upstream from CHST5. F: Chromosome with region B replaced by region A upstream from CHST6. G: Chromosome with one copy of CHST5 preceded by region A, a second copy of CHST5 preceded by region B, and one copy of CHST6 preceded by region B. H: Chromosome with CHST5 and region B deleted leaving only region A upstream from CHST6. 166 Molecular Vision 2006; 12:159-76 ©2006 Molecular Vision

TABLE 4. SUMMARY OF ALL CHST6 MUTATIONS FOUND IN MCD Total Number number of of families Change families per country Source ------No mutation detected (on one or both alleles) 17 6 USA [43]* 2 France [42] 9 India [2-4] Deletion upstream from coding region 7 4 USA * 2 Japan [33] 1 Italy [52] Replacement upstream from coding region 3 3 Japan [33] Deletion of ORF 5 2 USA * 2 India [2] 1 Japan [33] c.1A>T (Met1?) 5 4 USA * 1 Germany [48] c.[6G>A; 7C>A] (Trp2X + Leu3Met) 1 1 India [4] c.15_16delCG (Arg5fs) 1 1 India [2] c.15_16insATGCTGTGCG (Val6fs) 1 1 Iceland [40] c.16_40del (Val6fs) 2 2 India [3,4] c.44T>C (Leu15Pro) 1 1 France [42] c.51delG (Gln18fs) 1 1 USA * c.52C>T (Gln18X) 1 1 India [3] c.65T>G (Leu22Arg) 1 1 India [2] c.91C>T (Pro31Ser) 2 1 USA * 1 Britain [41] c.94_100del (Ser32fs) 1 1 India [3] c.124C>T (His42Tyr) 1 1 India [2] c.137T>C (Leu46Pro) 1 1 USA * c.148C>A (Arg50Cys) 1 1 Japan [33] c.148C>T (Arg50Cys) 2 2 India [2] c.149G>T (Arg50Leu) 2 2 India [2] c.152C>T (Ser51Leu) 2 1 USA [43] 1 Vietnam [49] c.155G>A (Gly52Asp) 2 2 India [3] c.158C>T (Ser53Leu) 8 1 USA * 7 India [2,3] c.161C>T (Ser54Phe) 1 1 India [4] c.[166G>A; 167T>G] (Val56Arg) 1 1 India [4] c.172C>T (Gln58X) 1 1 France [42] c.176T>C (Leu59Pro) 2 2 Vietnam [49] c.180delC (Phe60fs) 3 3 India [3,4] c.182A>C (Asn61Thr) 1 1 France [42] c.189C>G (His63Gln) 1 1 USA * c.196G>C (Val66Leu) 1 1 Vietnam [49] c.197T>A (Val66Asp) 1 1 Saudi Arabia [44] c.198delC (Phe67fs) 8 8 India [2,3] c.199T>A (Phe67Ile) 1 1 France [42] c.199T>G (Phe67Val) 1 1 Saudi Arabia [44] c.202T>C (Tyr68His) 1 1 France [42] c.213G>T (Glu71Asp) 1 1 Britain [41] c.214C>T (Pro72Ser) 2 1 USA [43] 1 Britain [41] c.217G>A (Ala73Thr) 1 1 India [4] c.217G>C (Ala73Pro) 1 1 USA * c.226G>A (Val76Met) 1 1 Vietnam [49] c.231G>A (Trp77X) 1 1 USA * c.231G>C (Trp77Cys) 1 1 USA [43] c.244C>T (Gln82X) 2 1 France [42] 1 Vietnam [45,49] c.271_273delGCTinsA (Ala91fs) 2 1 France [42] 1 USA * 167 Molecular Vision 2006; 12:159-76 ©2006 Molecular Vision

TABLE 4. CONTINUED. Total Number number of of families Change families per country Source ------c.274G>C (Val92Leu) 1 1 USA * c.277C>A (Arg93Ser) 3 3 USA [43]* c.278G>A (Arg93His) 1 1 India [2] c.290G>C (Arg97Pro) 1 1 India [2] c.[293C>G; 294C>G] (Ser98Trp) 1 1 India [3] c.[293C>T; 294C>G] (Ser98Leu) 1 1 India [4] c.304T>G (Cys102Gly) 4 2 USA [43] 1 France [42] 1 Italy [52] c.305G>A (Cys102Tyr) 2 2 India [2] c.310A>G (Met104Val) 1 1 USA [43] c.320T>C (Phe107Ser) 4 1 Britain [41] 3 India [3] c.329A>G (Tyr110Cys) 1 1 USA [43] c.340C>T (Arg114Cys) 1 1 USA * c.363C>G (Phe121Leu) 2 1 USA * 1 India [3] c.363_364insC (Gln122fs) 1 1 France [42] c.365A>C (Gln122Pro) 2 2 USA [43]* c.369G>A (Trp123X) 2 2 India [3,4] c.375_376insGGCCGTGC (Ser126fs) 3 3 Vietnam [45,49] c.379C>T (Arg127Cys) 4 1 Saudi Arabia [44] 3 India [2] c.383C>T (Ala128Val) 6 6 Iceland [40] c.391T>C (Ser131Pro) 2 1 France [42] 1 India [4] c.392C>T (Ser131Leu) 3 3 USA * c.414_415insTT (Pro139fs) 1 1 Japan [33] c.418C>T (Arg140X) 2 1 USA [50] 1 Britain [51] c.455T>C (Leu152Pro) 1 1 France [42] c.459C>A (Cys153X) 2 2 India [3,4] c.[494G>C; 495C>T] (Cys165Ser) 9 8 Saudi Arabia [44] 1 India [4] c.495C>G (Cys165Trp) 1 1 India [4] c.497G>C (Arg166Pro) 2 1 France [42] 1 Iceland [40] c.500C>T (Ser167Phe) 1 1 India [4] c.521A>G (Lys174Arg) 3 3 Japan [33] c.529C>T (Arg177Cys) 2 2 USA * c.530G>A (Arg177His) 1 1 Japan [47] c.533T>G (Phe178Cys) 1 1 India [4] c.545delA (Gln182fs) 13 13 India [2,4] c.573_574insC (Ala192fs) 1 1 USA * c.578T>C (Leu193Pro) 1 1 India [4] c.581_586delACCTACinsGGT 2 2 India [2] (Asn194_Arg196delinsArgCys) c.587_588insACG (Arg196_Ile197insArg) 1 1 India [3] c.593T>A (Val198Glu) 1 1 Italy [52] c.599T>G (Leu200Arg) 16 12 USA [43]* 2 Britain [41,51] 1 France [42] 1 Italy [52] c.604C>A (Arg202Ser) 2 2 India [3,4] c.607G>A (Asp203Asn) 2 2 USA * c.609C>A (Asp203Glu) 2 2 Japan [33] 168 Molecular Vision 2006; 12:159-76 ©2006 Molecular Vision

TABLE 4. CONTINUED. Total Number number of of families Change families per country Source ------c.611C>A (Pro204Gln) 3 1 France [42] 1 India [3] 1 Japan [47] c.611C>G (Pro204Arg) 1 1 India [4] c.612_614delGCGinsAT (Arg205fs) 2 2 India [2,3] c.614G>A (Arg205Gln) 2 2 India [2] c.614G>T (Arg205Leu) 1 1 Japan [47] c.616G>A (Ala206Thr) 1 1 India [2] c.617C>T (Ala206Val) 1 1 Britain [41] c.629C>T (Ser210Phe) 1 1 India [3] c.631C>T (Arg211Trp) 5 5 Japan [33,47] c.632G>A (Arg211Gln) 8 8 Vietnam [49] c.649G>A (Ala217Thr) 2 2 Japan [39,47] c.656_657insCTG (Ala219_Arg220insTrp) 6 6 India [3,4] c.661G>T (Asp221Tyr) 2 2 India [3,4] c.663C>G (Asp221Glu) 5 5 India [3,4] c.668G>A (Gly223Asp) 1 1 USA [50] c.[682A>G; 683C>A] (Thr228Asp) 1 1 USA * c.696G>A (Trp232X) 1 1 Vietnam [49] c.738C>G (Cys246Trp) 1 1 USA * c.740delG (Arg247fs) 1 1 USA * c.744C>G (Ser248Arg) 1 1 USA * c.746A>C (His249Pro) 1 1 India [2] c.803A>G (Tyr268Cys) 2 2 Vietnam [49] c.814C>A (Arg272Ser) 1 1 India [4] c.815G>A (Arg272His) 1 1 USA * c.820G>A (Glu274Lys) 2 1 India [2] 1 Japan [33] c.827T>C (Leu276Pro) 5 3 USA [43]* 1 Britain [51] 1 India [4] c.925G>T (Gly309X) 1 1 India [3] c.991C>T (Gln331X) 1 1 USA [43] c.993G>T (Gln331His) 2 1 USA [50] c.1000C>T (Arg334Cys) 4 3 USA * 1 India [4] c.1002_1012delinsTTG (His335fs) 2 2 India [4] c.1039G>T (Glu347X) 1 1 India [3] c.1046G>A (Cys349Tyr) 1 1 USA * c.1047C>G (Cys349Trp) 1 1 USA * c.1056_1078del (Leu353fs) 1 1 India [2] c.1060_1061insGTGCGCTG (Gln354fs) 1 1 USA [43] c.1072T>G (Tyr358Asp) 1 1 Britain [51] In the “change” column, mutations reported previously have been changed from the authors’ nomenclature to conform with recommended nomenclature. All previous reports have designated the first nucleotide of the CHST6 cDNA sequence as documented by Akama et al. [33] (GenBank accession number AF219990) as nucleotide +1, except for Gruenauer-Kloevekorn et al. [48], who designated the first nucleotide of the CHST6 cDNA sequence as reported by Hemmerich et al. [36] (GenBank accession number NM_021615) as nucleotide +1, and Sultana et al. [4], who designated the first nucleotide of the ATG initiation codon as nucleotide +1 (recommended nomenclature). An asterisk in the “Source” column indicates an inclusion of data from this article.

169 Molecular Vision 2006; 12:159-76 ©2006 Molecular Vision

CHST6 1 ------CHST5 1 ------CHST4 1 ------CHST7 1 ------MKGRRRRRREYCK CHST2 1 MSRSPQRALPPGALPRLLQAAPAAAPRALLPQWPRRPGRRWPASPLGMKVFRRKALVLCA CHST1 1 ------CHST3 1 ------MEKGLTLPQDCRDFVHSLKMRSK

hydrophobic site

______> | Changes ?XM << P CHST6 1 ------MWLPRVSSTAVTALL CHST5 1 ------MGMRARVP------KVAHSTRRPPAARMWLPRFSSKTVTVLL CHST4 1 ------MLLPKKMKLLLFLVSQ CHST7 14 FALLLVLYTLVLLLVP------SVLDGGRDGDKGAEHCPGLQRSLGVWSL CHST2 61 GYALLLVLTMLNLLDYKWHKEPLQQCNPDGPLGAAAGAAGGSWGRPGPPPAGPPRAHARL CHST1 1 ------MQCSWKAVLLLALASI CHST3 24 YALFLVFVVIVFVFIEKENKIISRVSDKLKQIPQALADANSTDPALILAENASLLSLSEL

hydrophobic site 5'-phosphate binding site ______| |L < C Changes X R S< Y P CL CHST6 16 LAQT------FLLLFLVSRPGPSSPAGGEARVHVLVLSSWRS CHST5 37 LAQTT------CLLLFIISRPGPSSPAGGEDRVHVLVLSSWRS CHST4 17 MAILA------LFFHMYSHNISSLSMKAQPERMHVLVLSSWRS CHST7 58 EAAAAGEREQGA-----EARAAEEGGANQSPRFPSNLSGAVGEAVSREKQHIYVHATWRT CHST2 121 DLRTPYRPPAAA-----VGAAPAAAAGMAGVAAPPGNGTRGTGGVGDKRQLVYVFTTWRS CHST1 17 AIQYTAIRTFTA-----KSFHTCPGLAEAGLAERLCEESPTFAYNLSRKTHILILATTRS CHST3 84 DSAFSQLQSRLRNLSLQLGVEPAMEAAGEEEEEQRKEEEPPRPAVAGPRRHVLLMATTRT

5'-phosphate binding site __ | < LI T X S W G Changes DLF R XP

N-glycosylation site ___ | | > P Changes V S C C LPX >CV L >X CHST6 104 MDVFDAYLPWR------RNLSDLFQWAVSRALCSPPACSAFPRG----AISSEAV CHST5 126 MDVFDAYMPQS------RNLSAFFNWATSRALCSPPACSAFPRG----TISKQDV CHST4 106 MSVFDAYMEPGP------RRQSSLFQWENSRALCSAPACDIIPQD----EIIPRAH CHST7 165 FSVLRLYAPPGDPAARAPDTANLTTAALFRWRTNKVICSPPLCPGAPRARAEVGLVEDTA CHST2 228 LSVFQLYSPAGSGGR------NLTTLGIFGAATNKVVCSSPLCPAYRKEVVG--LVDDRV CHST1 132 LYFLENYIKPPPVN------HTTDRIFRRGASRVLCSRPVCDPPGPAD---LVLEEGD CHST3 200 LYVLEHFITPLP------EDHLTQFMFRRGSSRSLCEDPVCTPFVKK-----VFEKYH

Figure 3. Contiues on the next page. 170 Molecular Vision 2006; 12:159-76 ©2006 Molecular Vision

3'-phosphate binding site ______| # S C NQQT Changes PX WPF R HC < >P±±±+E R SERLV CHST6 149 CKP-LCARQSFTLAREACRSYSHVVLKEVRFFNLQVLYPLLSDPALNLRIVHLVRDPRAV CHST5 171 CKT-LCTRQPFSLAREACRSYSHVVLKEVRFFNLQVLYPLLSDPALNLRIVHLVRDPRAV CHST4 152 CRL-LCSQQPFEVVEKACRSYSHVVLKEVRFFNLQSLYPLLKDPSLNLHIVHLVRDPRAV CHST7 225 CER-SCPPVAIRALEAECRKYPVVVIKDVRLLDLGVLVPLLRDPGLNLKVVQLFRDPRAV CHST2 280 CK--KCPPQRLARFEEECRKYRTLVIKGVRVFDVAVLAPLLRDPALDLKVIHLVRDPRAV CHST1 181 CVR-KCGLLNLTVAAEACRERSHVAIKTVRVPEVNDLRALVEDPRLNLKVIQLVRDPRGI CHST3 247 CKNRRCGPLNVTLAAEACRRKEHMALKAVRIRQLEFLQPLAEDPRLDLRVIQLVRDPRAV

3'-phosphate binding site N-glycosylation site ______|W Y | | Changes FQ T +E D D X CHST6 208 LRSREQTAKALARDNGIVLGTN------GTWVEADPGLRV CHST5 230 LRSREAAGPILARDNGIVLGTN------GKWVEADPHLRL CHST4 211 FRSRERTKGDLMIDSRIVMGQH------EQKLKKEDQPYY CHST7 284 HNSRLKSRQGLLRESIQVLRTRQRGDRFHRVLLAHGVGARPGGQSRALPAAPRADFFLTG CHST2 338 ASSRIRSRHGLIRESLQVVRSRDPRAHRMPFLEAAG-HKLG---AKKEGVGGPADYHALG CHST1 240 LASRSETFRDTYRLWRLWYGTG------RK—PYNLDVT CHST3 307 LASRMVAFAGKYKTWKKWLDDE------GQDGLREEEVQ

S Changes W

N-glycosylation site N-glycosylation site ______| | | | X Y Changes X H C# X W <> D CHST6 302 WIHNITHGSGPGARREAFKTSSRNALNVSQAWRHALPFAKIRRVQELCAGALQLLGYRPV CHST5 324 WIHNITHGSGIGKPIEAFHTSSRNARNVSQAWRHALPFTKILRVQEVCAGALQLLGYRPV CHST4 305 WVHNITRGKGMGD--HAFHTNARDALNVSQAWRWSLPYEKVSRLQKACGDAMNLLGYRHV CHST7 404 FALNMTRGAAYGAD-RPFHLSARDAREAVHAWRERLSREQVRQVEAACAPAMRLLAYPRS CHST2 454 FALNMTSGSGSSS--KPFVVSARNATQAANAWRTALTFQQIKQVEEFCYQPMAVLGYERV CHST1 331 WIQNNTRGDPTLG--KHKYGTVRNSAATAEKWRFRLSYDIVAFAQNACQQVLAQLGYKIA CHST3 400 WIQKNTQAAHDGS--G-IYSTQKNSSEQFEKWRFSMPFKLAQVVQAACGPAMRLFGYKLA

CHST6 362 YSEDEQRNLALDLVLPRGLNGFTWASSTASHPRN CHST5 384 YSADQQRDLTLDLVLPRGPDHFSWASPD------CHST4 363 RSEQEQRNLLLDLLS------T-WTVPEQIH--- CHST7 463 GEEGDAEQPREGETP------LEMDADGAT---- CHST2 512 NSPEEVKDLSKTLLR------KPRL----- CHST1 389 ASEEELKNPSVSLVE------ERDFRPFS----- CHST3 457 RDAAALTNRSVSLLE------ERGTFWVT-----

Figure 3. Alignment of CHST6 mutations with amino acid sequences of carbohydrate sulfotransferases. Shown here is the alignment of CHST6 mutations with amino acid sequences of human galactose/N-acetylgalactosamine/-N-acetylglucosamine 6-O-sulfotransferase family of carbohydrate sulfotransferases (CHST1-7). In the figure, the red colored residues are identical to the consensus sequence and the blue colored residues are similar to the consensus sequence.The hydrophobic site, 5'-phosphate binding site, 3'-phosphate binding site and the N- glycosylation sites of CHST6 are also shown. The less than sign (<) indicates a deletion with frameshift, the greater than sign (>) indicates an insertion with frameshift, the sharp (#) indicates an insertion plus deletion with frameshift, the plus sign (+) indicates an in-frame insertion, and the plus/minus sign (±) indicates an in-frame insertion plus deletion. 171 Molecular Vision 2006; 12:159-76 ©2006 Molecular Vision other family (family 24), neither sequence analysis of the cod- insertion upstream of this gene. In family 27, there was insuf- ing region of CHST6 nor a screening of the upstream region ficient DNA to determine whether or not there were upstream using the method of Akama et al. [33] disclosed mutations deletions or insertions. DNA was not available in two sub- capable of explaining the genetic basis for MCD. In one other jects with MCD type II but one allele in these cases could be family (family 16), there was a heterozygous upstream dele- determined by analyzing DNA from children of the subjects. tion and a heterozygous coding region mutation, but it could The nucleotide changes in CHST6 in each of these carriers not be determined if they were or were not on the same chro- were c.392C>T (Ser131Leu), c681C>T (Gly227Gly), mosome. In one family (family 27) there were no coding re- c.682A>G, 683C>A (Thr228Asp; family 30), and c.607G>A gion mutations and there was insufficient DNA to determine (Asp203Asn; family 31). In 14 families (19 affecteds and 23 whether or not upstream deletions or replacements were carriers), the MCD immunophenotype was not determined. present. In three other families (families 29, 30, and 31), DNA could not be obtained from affected family members, and in DISCUSSION each of these families, only one chromosome-carrying muta- Eighteen of the mutations found in this investigation have tion could be detected among family members who were car- apparently not been reported before (c.51delG [Gln18fs], riers. c.137T>C [Leu46Pro], c.189C>G [His63Gln], c.217G>C Heterozygous mutations were detected in exon 3 of [Ala73Pro], c.231G>A [Trp77X], c.274G>C [Val92Leu], CHST6 in 18 families (families 1, 3, 4, 6, 7, 9, 10, 13, 15, 16, c.340C>T [Arg114Cys], c.392C>T [Ser131Leu], c.529C>T 17, 19, 20, 22, 23, 25, 26, and 28) and almost all were in asso- [Arg177Cys], c.573_574insC [Ala192fs], c.607G>A ciation with another heterozygous mutation in the coding re- [Asp203Asn], c.682A>G, 683C>A [Thr228Asp], c.738C>G gion of CHST6 on the other chromosome. The three excep- [Cys246Trp], c.740delG [Arg247fs], c.744C>G [Ser248Arg], tions had an upstream deletion (families 1, 16, and 19) on the c.815G>A [Arg272His], c.1046G>A [Cys349Tyr], and other chromosome. In 14 instances, two or more independent c.1047C>G [Cys349Trp]), but 12 have been previously docu- heterozygous mutations were present. Aside from having two mented. They include c.1A>T (Met1?) [48], c.91C>T heterozygous nucleotide changes in CHST6 (c.484C>G, (Pro31Ser) [41] c.158C>T (Ser53Leu) [2,3], Arg162Gly and c.599T>G, Leu200Arg), family 9 had another c.271_273delGCTinsA (Ala91fs) [42], c.277C>A (Arg93Ser) homozygous missense mutation (c.1000C>T, Arg334Cys). [43], c.363C>G (Phe121Leu) [3], c.365A>C (Gln122Pro) [43], Family 23 had two heterozygous mutations, but it was not c.599T>G (Leu200Arg) [41-43,51,52], c.827T>C (Leu276Pro) possible to establish whether they were or were not on the [43], c.1000C>T (Arg334Cys) [4], upstream deletions [33,52], same chromosome because of insufficient DNA. In family 1, and a deletion of the coding region [2,33]. Together with past one subject (case 1) had a homozygous c.365A>C (Gln122Pro) reported genomic DNA analyses, this brings the number of mutation, whereas an affected sibling (case 2) had a heterozy- identified CHST6 mutations in MCD to 124 (Table 4). This gous c.365A>C(Gln12Pro) mutation together with a deletion vast number underscores the marked allelic heterogeneity in upstream of CHST6 on the other chromosome 16. CHST6 that has been previously documented in subjects with Homozygous mutations were detected in the coding re- MCD in Britain [41], France [42], Iceland [40], India [2-4], gion of CHST6 in nine families (families 1, 2, 5, 8, 9, 12, 14, Italy [52], Japan [33,47], Saudi Arabia [44], United States 18, and 21), and in two of them (families 5 and 18) a major [43,46,50], and Vietnam [45,49]. The vast majority of sub- portion of CHST6 that included the ORF was deleted. An- jects with MCD have missense and nonsense mutations in other family (family 11) had a homozygous deletion upstream CHST6 that involve a single nucleotide change that is pre- from CHST6. One family (family 9) had a homozygous dicted to alter a conserved amino acid [2-4,33,39-45,47-52]. c.1000C>T (Arg334Cys) mutation, but also two heterozygous That these nucleotide changes are disease related is supported mutations (c.484C>G, Arg162Gly; c.599T>G, Leu200Arg). by the fact that they have not been detected in analyses of In another family (family 1), one affected individual was ho- CHST6 in numerous control subjects in different parts of the mozygous for a single mutation (c. 365A>C, Gln122Pro), world [2-4,33,40-45,47,49,52]. In an earlier study of Icelan- while another affected individual was heterozygous for this dic individuals, c.383C>T (Ala128Val) was detected in four mutation, but also had a deletion upstream of CHST6 on the of 50 healthy controls. However, it was most likely a mis- other chromosome. Whereas homozygous mutations are ex- sense mutation occurring in carriers, because it was associ- pected in the offspring of inbred matings, we were only able ated with MCD when present in a homozygous state [40]. to establish consanguinity in two families with extensive ge- Other MCD-causing mutations are nucleotide insertions nealogical analyses. or deletions in the coding region of CHST6 that cause frame- Results related to the MCD type: Most subjects studied shift changes and a few deletions or substitutions upstream of had MCD type I (26 patients from 13 families). Six affected CHST6. The latter is a consequence of the marked sequence individuals with MCD type II were from four families (fami- similarity of nucleotide sequences within the tandem CHST5 lies 24, 27, 30, and 31). The c.231G>A mutation that gener- and CHST6 genes and the adjacent regions. It predisposes the ated a stop codon (Trp77X) was found in three subjects with region to anomalous chromosomal crossovers that lead to de- MCD type 1. Affected subjects in two families (families 24 letions and replacements within the involved chromosomes and 27) with MCD type II lacked mutations in the coding re- (Figure 2). Such defects in the promoter region were detected gion of CHST6, and those in family 24 also had no deletion or in six subjects with MCD in the present study and in 12 previ- 172 Molecular Vision 2006; 12:159-76 ©2006 Molecular Vision ously documented cases [33,52]. Fifty-five of the documented c.484C>G; 599T>G (Arg162Gly and Leu200Arg) [43,52]. CHST6 mutations have been detected in more than one family Together, these observations suggest that this part of human with MCD; some of these families may be related to each chromosome 16 may be prone to mutations that may not be other as a common ancestor has not been excluded yet. readily corrected by the usual DNA repair mechanisms. Our detection of four SNPs in CHST6 that did not affect One case of MCD in this study could not be explained by an amino acid (c.258A>C [Ala86Ala], c.294C>G [Ser98Ser], mutations in the coding region of CHST6, by major deletions c.465G>A [Arg155Arg], and c.681C>T [Gly227Gly]) together or insertions in the upstream region, or by splice site muta- with other reported SNPs brings the number of identified SNPs tions which create or destroy signals for exon-intron splicing in CHST6 to 88. SNPs outside of the coding region could af- [68]. Others have also failed to detect mutations on one or fect gene expression by altering or activating splice sites or both alleles of individuals with MCD [2-4,42,43]. A genetic by affecting the stability or translational efficiency of the tran- explanation for these cases of MCD remains unknown, but script [64], but in individuals in whom we found these SNPs several theoretical possibilities exist. It is conceivable that the the MCD could be explained by other mutations. Although relevant abnormality involves transcription factor binding sites SNPs in the 3'-UTR have been reported to affect expression (regulatory promoters) in the region immediately upstream of [65] and mRNA stability [66], the ones we detected the minimal promoter that direct the expression of the gene or (c.3342A>T and c.3501G>A) were also homozygous in some cis-acting distant genomic elements (enhancers, repressors, unaffected family members, indicating that they are not in- or insulators) located upstream or downstream of the transcrip- volved in the pathogenesis of MCD. tion unit [69]. In this regard, Hemmerich et al. [36], suggested As illustrated in Figure 3, mutations found in MCD have that the closely proximated CHST5 and CHST6 genes may be been in highly conserved parts of carbohydrate 6-O- regulated by common promoters and/or enhancers, and they sulfotransferases, suggesting that the altered amino acids im- drew attention to a conspicuous triplet of binding sites for the pair the normal functioning of the encoded enzyme and are zinc-dependent Sp1 transcription factor upstream of CHST6, pathogenic. Seventeen mutations involve the domains neces- which has three contiguous Zn (II) fingermotifs believed to sary for the interaction between the sulfotransferase and the be metalloprotein structures that interact with DNA. The Sp1 sulfate donor PAPS [35]. Six of these mutations are situated binding sites are present in the 5'-regulatory sequences of nu- within the 5'-PSB (5'-phosphosulfate-binding) domain that merous genes that encode carbohydrate-modifying enzymes interacts with the 5'-phosphate group of PAPS [67], and 11 [70]. In other protein-coding genes, a 5'-promoter element mutations involve the 3'-phosphate-binding domain that in- contiguous with the transcription site (the minimal or core teracts with the 3'-phosphate of PAPS. There are also four promoter) of the gene is necessary to splice the exons needed mutations in the hydrophobic site, but none in any of the four to produce the mRNA that encodes the appropriate protein glycosylation sites. [71]. This is presumably also true for CHST6. Other yet to be All SNPs in the coding region of CHST6 that alter a codon identified regulatory elements of CHST6 may also play a role, are presumed to be of pathogenic significance because a con- and genes on other chromosomes may affect the gene expres- served amino acid is altered. However, at least one SNP sion as well. A defect may reside in the 5'-untranslated region (c.484C>G), which is predicted to change arginine to glycine (5'-UTR) of CHST6, which has not been fully analyzed, be- at codon 162, does not seem to alter the encoded cause this part of other genes contributes to the specificity sulfotransferase significantly. This SNP has been previously and overall efficiency of translation initiation [72]. Moreover, reported in 6.2% of an American control sample [43] and in genetic heterogeneity remains to be excluded. 9.4% of Italian controls [52]. We have also observed it in 12.0% In their landmark publication, Akama et al. [33] provided of an American control sample and in 16.2% of an Icelandic evidence that MCD type II might be caused by genetic abnor- control population (unpublished). Moreover, Abbruzzese et malities upstream of CHST6. While a defect in the promoter al. [52] suggested that this amino acid change does not affect region of CHST6 is expected to control corneal sulfotransferase enzyme activity since it is not conserved in mouse intestinal activity and hence might cause the milder immunophenotype GlcNAc 6-O-sulfotransferase which has been shown to have of MCD characterized by AgKS in the serum and cornea, other the same enzymatic activity as human corneal GlcNAc 6-O- cases of MCD type II have not only had other mutations in the sulfotransferase [39]. coding region of CHST6, but have also lacked evidence of a Aside from the marked allelic heterogeneity of CHST6 in defect upstream of CHST6. A possible molecular explanation MCD, it is noteworthy that 10 families (families 6, 9, 12, 13, for the different immunophenotypes of MCD was provided 15, 22, 23, 26, 28, and 30) with MCD in the present study had by an unusual sibship that contained both MCD types I and II two SNPs in the coding region of CHST6 in a single chromo- [30,50]. In that family, the sibling with the greater deficiency some. In addition to our study, other cis SNP combinations of AgKS (MCD type I) had homozygous c.418C>T (Arg140X) have included c.6G>A; 7C>A (Trp2X and Leu3Met) [4], mutations in CHST6, which is predicted to generate a stop- c.166G>A; 167T>G; 500C>T (Val56Arg and Ser167Phe) [4], codon and hence a truncated sulfotransferase. The individuals c.213G>T; 214C>T (Glu71Asp and Pro72Ser) [41], with the milder MCD type II in the sibship were heterozygous c.293C>G; 294C>G (Ser98Trp) [3], c.293C>T; 294C>G for c.418C>T and for cis c.993G>T and c.668G>A nucleotide (Ser98Leu) [4], c.494G>C; 495C>T (Cys165Ser) [4,44], changes in CHST6 and would presumably still have some re- c.668G>A; 993G>T (Gly223Asp and Gln331His) [50], and sidual enzymatic function albeit defective. It is noteworthy 173 Molecular Vision 2006; 12:159-76 ©2006 Molecular Vision that the one family in the present study with a heterozygous 7. Jonasson F, Johannsson JH, Garner A, Rice NS. Macular corneal stop codon in the coding region of CHST6 (family 4) had MCD dystrophy in Iceland. Eye 1989; 3:446-54. type I. However, the molecular basis for the different 8. Ehlers N, Bramsen T. Central thickness in corneal disorders. Acta immunophenotypes is clearly more complex as a molecular Ophthalmol (Copenh) 1978; 56:412-6. 9. Donnenfeld ED, Cohen EJ, Ingraham HJ, Poleski SA, Goldsmith genetic study of MCD in Saudi Arabia found identical CHST6 E, Laibson PR. Corneal thinning in macular corneal dystrophy. mutations in families with MCD types I, IA, and II [44]. Am J Ophthalmol 1986; 101:112-3. Despite overwhelming evidence implicating CHST6 in 10. Quantock AJ, Meek KM, Ridgway AE, Bron AJ, Thonar EJ. the pathogenesis of MCD, an understanding of the molecular Macular corneal dystrophy: reduction in both corneal thickness events that lead to the characteristic lesions of MCD remain and collagen interfibrillar spacing. Curr Eye Res 1990; 9:393- incomplete. The intracellular storage of GAGs may reflect a 8. failure of transport of PGs with insufficiently sulfated 11. Bücklers M. Die erblichen Hornhautdystrophien: Dystrophiae lactosaminoglycans from the Golgi apparatus, but the charac- corneae hereditariae. Stuttgart, Ferdinand Enke; 1938. teristic extracellular deposits in Descemet’s membrane remains 12. Jones ST, Zimmerman LE. Macular dystrophy of the cornea (Groenouw type II); clinicopathologic report of two cases with to be explained. Also, a knockout of CHST6 might be expected comments concerning its differential diagnosis from lattice dys- to cause MCD, but mice and other nonprimates lack CHST6 trophy (Biber-Haab-Dimmer). Am J Ophthalmol 1959; 47:1- and can be viewed as being equivalent to animals in which 16. CHST6 has been deleted by genetic engineering yet they do 13. Jones ST, Zimmerman LE. Histopathologic differentiation of not manifest MCD. However, the KS in corneas of mice are granular, macular and lattice dystrophies of the cornea. Am J predominantly undersulfated and express a low level of reac- Ophthalmol 1961; 51:394-410. tivity with the 5D4 antibody used to differentiate MCD types 14. Klintworth GK, Meyer R, Dennis R, Hewitt AT, Stock EL, Lenz I and II. The epitope of 5D4 is linear pentasulfated sequences ME, Hassell JR, Stark WJ Jr, Kuettner KE, Thonar EJ. 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The print version of this article was created on 10 Mar 2006. This reflects all typographical corrections and errata to the article through that date. Details of any changes may be found in the online version of the article. α 176