Combined use of multiplex ligation-dependent probe amplification and automatic sequencing for identification of KAL1 defects in patients with Kallmann syndrome

ObjectiveTo investigate the role of KAL1 abnormalities in Brazilian patients with Kallmann syndrome.DesignIn vitro experiments.SettingAcademic medical center.Patient(s)One hundred fifteen Brazilian patients (98 men) with Kallmann syndrome.Intervention(s)Peripheral blood leukocytes were used to obtain DNA.Main Outcome Measure(s)Direct sequencing and multiplex ligation-dependent probe amplification were used to identify KAL1 abnormalities.Result(s)We identified four KAL1 mutations (p.Met1?, p.Ala33Glyfs, p.Arg257*, and p.Trp462*) and two multiple exon deletions (exons 1–2 and 3–14) in six new male patients. Overall, 17 KAL1 defects (14.8%) were identified in the entire cohort of patients with Kallmann syndrome, including previously studied cases. KAL1-mutated patients presented with a more severe reproductive and nonreproductive phenotype (synkinesia, renal malformations, cryptorchidism, and anatomic olfactory abnormalities) in comparison with patients without KAL1 mutations. Intragenic deletions were one of the most often encountered defects (29.4%). These deletions can be missed by polymerase chain reaction (PCR) due to Yq11.2 KAL1 pseudogene (KALP) spurious amplification.Conclusion(s)These results indicate that intragenic multiexon deletions are one of the most frequent KAL1 abnormalities, which can be more accurately detected by multiplex ligation-dependent probe amplification. In addition, KAL1 sequencing results should be interpreted with caution, and stringency conditions of the PCR reaction should be adjusted to avoid pseudogene amplification.