Investigating the “Dark” Genome: First Report of Partington Syndrome in Cyprus

<b>Background/Objectives</b>: X-linked intellectual disability (XLID) is a highly heterogeneous disorder accounting for ~10% of all males with ID. Next-generation sequencing (NGS) has revolutionized the discovery of causal XLID genes and variants; however, many cases remain unresolved. We present a four-generation syndromic XLID family with multiple males exhibiting variable degree of ID, focal dystonia and epilepsy. <b>Methods</b>: Extensive cytogenetic and targeted genetic testing was initially performed, followed by whole-exome sequencing (WES) and short-read whole-genome sequencing (WGS). Apart from the routine NGS analysis pipelines, sequencing data was revisited by focusing on poorly covered/mapped regions on chromosome X (chrX), to potentially reveal unidentified clinically relevant variants. Candidate variant validation and family segregation analysis were performed with Sanger sequencing. <b>Results</b>: All initial diagnostic testing was negative. Subsequently, 300 previously reported "dark" chrX coding DNA sequences, overlapping 97 genes, were cross-checked against 29 chrX genes highly associated (<i>p</i> < 0.05) with ID and focal dystonia, according to Phenomizer. Manual inspection of the existing NGS data in two low-coverage regions, chrX:25013469-25013696 and chrX:111744737-111744820 (hg38), revealed a recurrent pathogenic <i>ARX</i> variant NM_139058.3:c.441_464dup p.(Ala148_Ala155dup) (ARXdup24) associated with non-syndromic or syndromic XLID, including Partington syndrome. Sanger sequencing confirmed ARXdup24 in all affected males, with carrier status in their unaffected mothers, and absence in other unaffected relatives. <b>Conclusions</b>: After several years of diagnostic odyssey, the pathogenic ARXdup24 variant was unmasked, supporting a genotype-phenotype correlation in the first Partington syndrome family in Cyprus. This study highlights that re-examining underrepresented genomic regions and using phenotype-driven tools can provide critical diagnostic insights in unresolved XLID cases.