title: Combined use of multiplex ligation-dependent probe amplification and automatic sequencing for identification of KAL1 defects in patients with Kallmann syndrome.
abstract: OBJECTIVE
  To investigate the role of KAL1 abnormalities in Brazilian patients with Kallmann syndrome.
  
  
  METHODS
  In vitro experiments.
  
  
  METHODS
  Academic medical center.
  
  
  METHODS
  One hundred fifteen Brazilian patients (98 men) with Kallmann syndrome.
  
  
  METHODS
  Peripheral blood leukocytes were used to obtain DNA.
  
  
  METHODS
  Direct sequencing and multiplex ligation-dependent probe amplification were used to identify KAL1 abnormalities.
  
  
  RESULTS
  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.
  
  
  CONCLUSIONS
  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.
authors.affiliation: Disciplina de Endocrinologia e Metabologia, Laboratório de Hormônios e Biologia Molecular, University of São Paulo, São Paulo, Brazil.
authors.forename: Luciana Ribeiro
authors.initials: LR
authors.lastname: Montenegro
authors.forename: Leticia F G
authors.initials: LF
authors.lastname: Silveira
authors.forename: Cintia
authors.initials: C
authors.lastname: Tusset
authors.forename: Margaret
authors.initials: M
authors.lastname: de Castro
authors.forename: Beatriz R
authors.initials: BR
authors.lastname: Versiani
authors.forename: Ana Claudia
authors.initials: AC
authors.lastname: Latronico
authors.forename: Berenice Bilharinho
authors.initials: BB
authors.lastname: Mendonca
authors.forename: Ericka B
authors.initials: EB
authors.lastname: Trarbach
mesh_terms: D000328:Adult; D001331:Automation; D001483:Base Sequence; D004252:DNA Mutational Analysis / Q000295:instrumentation / Q000379:methods*; D016326:Extracellular Matrix Proteins / Q000235:genetics*; D005260:Female; D005787:Gene Frequency; D059014:High-Throughput Nucleotide Sequencing / Q000295:instrumentation / Q000379:methods*; D006801:Humans; D017436:Kallmann Syndrome / Q000175:diagnosis / Q000453:epidemiology / Q000235:genetics*; D008297:Male; D060885:Multiplex Polymerase Chain Reaction; D009154:Mutation / Q000235:genetics / Q000502:physiology; D009419:Nerve Tissue Proteins / Q000235:genetics*; D015995:Prevalence; D011544:Pseudogenes / Q000235:genetics
chemical_list: C076266:ANOS1 protein, human; D016326:Extracellular Matrix Proteins; D009419:Nerve Tissue Proteins
keywords: KAL1 abnormalities; KALP spurious amplification; Kallmann syndrome; MLPA