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