Balkan Journal of Medical Genetics

ASSOCIATION OF VARIANTS IN THE CP, ATOX1 AND COMMD1 GENES WITH WILSON DISEASE SYMPTOMS IN LATVIA
Zarina A1,2*, Tolmane I3,4, Krumina Z2, Tutane AI1, Gailite L1
*Corresponding Author: Ms. Agnese Zarina, Scientific Laboratory of Molecular Genetics, Rīga Stradiņš University, 16 Dzirciema Street, Riga, Latvia, LV-1007. Tel.: +371-6706-1542. Fax: +371-67-471-815. E-mail: agnese.zarina@rsu.lv or zarina.agnese@gmail.com
page: 37

MATERIALS AND METHODS

This study protocol was approved by the Latvian Central Medical Ethics Comittee and was performed according to the Declaration of Helsinki. Informed consent was obtained from all patients. In the study, 64 unrelated patients with confirmed WD, were enrolled on the basis of Leipzig’s diagnostic criteria [11]. All patients included in the study carried at least four points according to the WD scoring system. According to the initial symptoms, WD patients were categorized in the following groups, as described elsewhere [12]: asymptomatic, hepatic, neurological/ psychiatric and neurological/hepatic. Genomic DNA was extracted from peripheral blood by a standard phenol/chloroform method. Allelic variant c.3207 C>A; p.H1069Q (rs76151636) was tested by polymerase chain reaction (PCR)-BiPASA (bidirectional PCR of specific alleles) [13]. Direct DNA sequencing of the ATP7B gene (promoter, exons and exon-intron boundaries) was performed for the patients with WD symptoms, being either heterozygous for H1069Q or without it on any allele. Methods for WD molecular confirmation and the group of WD patients have been described before in detail [14,15]. Direct Sequences of the ATOX1, COMMD1 and CP Genes. For sequencing of the CP gene promoter region, ATOX1 and COMMD1 gene exons and adjacent introns, primers selected using the open access Primer 3 program (available at http://bioninfo.ut.ee/primer3-0.4.0/) and previously reported primers were used [6,8]. For the genetic variants nomenclature, traditional names were used (if existing), or HGVS nomenclature according to reference sequences: CP gene (NM_000096.3, NP_000087.1); ATOX1 gene (NM_004045.3, NP_004036.1); COMMD1 gene (NM_152516.3, NP_689729.1). Allele frequencies in the European population were obtained from GnomAD database (https:/gnomad.broadinstitute.org/) [16]. For Linkage data, the 1000 Genome browser (https://www. international genome.org) was used [17]. If a variant was not reported to have a functional effect, it was characterized according to the American College of Medical Genetics and Genomics (ACMG guidelines [18], including in silico tools. The CP gene promoter region (not all of the gene) sequencing was selected based on information in other studies concluding that changes in the CP gene promoter may be associated with altered levels of ceruloplasmin [8]. Statistical Analyses. Data processing was performed using the Statistical Package for the Social Sciences (IBM SPSS®) version 22 (https://www.ibm.com/SPSS-Statistics/ Software), and PLINK 1.07 (http://zzz.bwh.harvard. edu/plink/). The comparison between patient groups was done by Fisher exact t-test. The frequencies of genotypes in the WD patient population before further data analysis were tested for Hardy-Weinberg equilibrium. The association of the COMMD1 and CP gene variants with WD phenotype was analyzed in the allelic, dominant, recessive and genotypic inheritance model.

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