Analysis of DPY19L2
(A) Schematic representation of DPY19L2 flanked by two LCRs, LCR1 and LCR2. LCR1 is located upstream of the gene and LCR2 downstream. Different sequences were amplified on both sides of the gene in order to determine the breakpoints: “a,” “b,” and “c” sequences are localized in LCR1, whereas “d” and “e” sequences are localized in LCR2. Mismatches between the two LCRs sequences are represented by (∗) in the 5′ side and by (.) in the 3′ side.
(B) PCR results of DPY19L2 exons and flanking regions. Patients Globo1, Globo7, Globo17, and Globo18 are deleted for exons 2, 7, 9, 10, 13, 17, and 21, whereas the positive control showed an amplification of all of these exons. All patients showed specific amplifications for region “a” in LCR1 and region “e” in LCR2. Globo1 and Globo18 are missing region “b” in LCR1, which is not the case for Globo7 and Globo17. Globo7 is the only patient missing region “d” in LCR2. A DNA ladder was included on both sides of each gel for accurate determination of the PCR band size.
(C) PCR of DPY19L2 breakpoints. This PCR was performed with the use of the “LCR1a forward” and “DPY19L2-BP reverse” primers (). The four globozoospermic patients showed a fragment of 1700 bp. The fertile brother (B1, IV-5 in A) is homozygous wild-type, because no fragment was amplified, whereas parents of Globo18 (P1 and P2) and Globo17 (P3 and P4) are heterozygous. A fertile man (positive control) was used as control of the PCR specificity. Amplification of exon 10 (lower panel) was used to control the presence or absence of DPY19L2.
(D) Table summarizing the PCR results of the four patients: (+) indicates presence of a PCR band at the corresponding size, and (-) indicates absence of a PCR band.
(E) Schematic representation of the breakpoint area determined for Globo1, Globo18, and the paternal allele of Globo17. The breakpoint area (DPY19L2-BP1) identified by PCR and sequencing is defined between the two discontinued lines and represented by a hatched box in the recombined LCR.
(F) Schematic representation of the homologous recombination that occurred in Globo7 and the maternal allele of Globo17. The breakpoint area (DPY19L2-BP2) identified by PCR and sequencing is defined between the two discontinued lines and represented by a hatched box in the recombined LCR.