Classical fragile-X phenotype in a female infant disclosed by comprehensive genomic studies

Abstract

Background: We describe a female infant with Fragile-X syndrome, with a fully expanded FMR1 allele and preferential inactivation of the homologous X-chromosome carrying a de novo deletion. This unusual and rare case demonstrates the importance of a detailed genomic approach, the absence of which could be misguiding, and calls for reflection on the current clinical and diagnostic workup for developmental disabilities.

Case presentation: We present a female infant, referred for genetic testing due to psychomotor developmental delay without specific dysmorphic features or relevant family history. FMR1 mutation screening revealed a methylated full mutation and a normal but inactive FMR1 allele, which led to further investigation. Complete skewing of X-chromosome inactivation towards the paternally-inherited normal-sized FMR1 allele was found. No pathogenic variants were identified in the XIST promoter. Microarray analysis revealed a 439 kb deletion at Xq28, in a region known to be associated with extreme skewing of X-chromosome inactivation.

Conclusions: Overall results enable us to conclude that the developmental delay is the cumulative result of a methylated FMR1 full mutation on the active X-chromosome and the inactivation of the other homologue carrying the de novo 439 kb deletion. Our findings should be taken into consideration in future guidelines for the diagnostic workup on the diagnosis of intellectual disabilities, particularly in female infant cases.

Keywords: Developmental disabilities in females; FMR1 methylated full mutation; Fragile-X syndrome; Skewing of X-chromosome inactivation; Xq28 deletion.

Fig. 1

Pedigree and summary of overall laboratory findings. a – Proband’s pedigree (III:1) showing FMR1 genotyping and XCI ratio results. b – Southern blot analysis using GLFXDig1 probe (Gene Link^TM , Hawthorne, NY, US) and DNA Molecular Weight Markers II and III, DIG-labeled from Merck KGaA, Darmstadt, Germany; Female control with a full mutation (C+) c.-128_-126[30];[250_400]; Proband III:1 showing complete absence of normal, active FMR1 allele. Premutation carriers (II:3 and II:4) show four fragments corresponding to the normal active, expanded active (2.8 kb and above) and normal inactive and expanded inactive (5.2 kb and above) alleles [21]. c - HUMARA results obtained in proband’s leukocytes (III:1) [10]. HhaI completely digested the maternal allele indicating that the other allele is fully methylated (ME) and suggesting total skewing of the XCI pattern. The additional two females tested (II:3 and I:4) showed a normal XCI pattern (data not shown). d – Array Comparative Genomic Hybridisation (aCGH) performed using the Cytochip ISCA 8x60K (Cambridge Bluegnome, Illumina Inc., San Diego CA,USA) showed a deletion of 439Kb in chromosome X within band Xq28, classified as pathogenic, involving the genes: F8; CTD-2183H9.7; EEF1A1P31; CTD- 2183H9.3; FUNDC2; CMC4; MTCP1; BRCC3; RP11-143H17.1; VBP1; RP13-228 J13.9; RAB39B; CLIC2; RP13-228 J13.6; RP13-228 J13.10. Array design included a median resolution of 120Kb throughout the genome (Backbone) with increased density oligonucleotide probes in selected regions associated with clinically relevant phenotypes, in line with the International Standard Cytogenetic Array (ISCA) consortium. Analysis was performed using the Bluefuse software (Cambridge Bluegnome, Illumina Inc., San Diego CA, US) coupled with publicly available databases. Mutation nomenclature guidelines suggested by the Human Genome Variation Society (HGVS) (http://varnomen.hgvs.org/) were used. FMR1 reference sequence NM_002024.5