idiopathic hypogonadotropic hypogonadism ( ihh ) is comprised of absent puberty , infertility and low serum gonadotrophins in the absence of a pituitary tumor .
ihh is due to impaired gonadotrophin releasing hormone ( gnrh ) release / action or gonadotrophin secretion .
ks appears to be the result of impairment of gnrh and olfactory neuron migration from the olfactory placode to the hypothalamus ( bhagavath and layman , 2007 ) .
mutations in genes involved in gnrh neuron migration ( kal1 and fgfr1 ) , as well as those expressed in either the hypothalamus or pituitary , including gnrhr , nrob1 , gpr54 , lep , lepr and pcsk1 account for the molecular etiology of 1520% of all ihh / ks patients ( hardelin , 2001 ; de roux , 2005 ; pitteloud et al . , 2006 ; bhagavath and layman , 2007 ; seminara , 2007 ) .
kal1 , gnrhr and fgfr1account for the majority of causative mutations . the molecular basis for most ihh / ks cases
digenic disease has been reported in two families with fgfr1 and either a gnrhr or a nelf mutation ( pitteloud et al . , 2007 ) .
most mutations identified in genetic diseases , including ihh / ks , are point mutations and small deletions / duplications detected by pcr - based dna sequencing ( kim et al . , 2008 ) .
pcr based sequencing may not detect heterozygous gene deletions , however , since the remaining normal allele will be amplified . therefore , it is possible that a substantial number of mutations remain undiagnosed .
multiplex ligation - dependent probe amplification ( mlpa ) is a relatively new method for detecting copy number variations in genomic sequences ( schouten et al . , 2002 ) .
mlpa involves overnight hybridization of adjacent primer pairs to the individual exons of multiple target genes simultaneously , followed by a ligation reaction of the probe pairs , which then serve as a template for multiplex pcr .
stuffer sequences that produce different sized fragments for resolution based upon size ( schouten et al . ,
since all probes have identical primer binding sites , only a single primer pair is required for multiplex pcr of all genes being studied .
heterozygous intragenic deletions have been found in 1520% of patients with cystic fibrosis ( audrezet et al . , 2004 ) ,
deafness ( del castillo et al . , 2003 ) and breast / ovarian cancer ( hogervorst et al . , 2003 ) who did not have point mutations in the corresponding genes .
we hypothesized that a similar percentage of ihh / ks patients would have heterozygous deletions of autosomal genes ( fgfr1 , gnrh1 , gnrhr , gpr54 or nelf ) and/or hemizygous kal1 gene deletions , deletions which would be missed by the traditional pcr - based sequencing methods .
additionally , since kal1 has a pseudogene homolog on the y chromosome , this may complicate the dna sequencing analysis ( del castillo et al . , 1992 ) .
one - hundred and twelve probands with ihh / ks were studied by mlpa ( table  i ) .
absent / impaired puberty by age 17 in girls and age 18 in boys , low serum gonadotrophins , no evidence of a pituitary tumor and otherwise normal pituitary function ( crowley et al . , 1985 ; bhagavath et al . ,
all males had total testosterone < 100 ng / dl ( normal 3001100 ng / dl ) , and all females had hypoestrogenic amenorrhea .
complete ihh was defined as absent breast development in females and testicular size 3 ml in males ( bhagavath et al . , 2006 ) .
incomplete ihh suggested the presence of prior steroid production and was defined as breast development of tanner 2 in females and testis size > 3 ml in males .
patient characteristics are shown in table  i. the medical college of georgia human assurance committee approved this study ; each patient signed informed consent .
fifty - four patients were studied for the presence of kal1 deletions ( kal1 kit ) and 100 ihh / ks patients were studied for deletions of an autosomal panel of fgfr1 , gnrh1 , gnrhr , gpr54 and nelf genes ( kal2 kit ) .
we estimated that we needed a sample size of 50 for study of kal1 gene deletions , since the prevalence of mutations is about 6% in anosmic males ( bhagavath et al . , 2007 ) .
the sample size for the autosomal gene panel ( kal2 kit ) was estimated to be 100 .
the prevalence of fgfr1 mutations in both normosmic ihh and ks is about 10% ( pitteloud et al . , 2006 ) , whereas gnrhr mutations occur in 34% of normosmic ihh ( bhagavath et al . , 2005 ) .
the prevalence of gnrh1 , nelf and gpr54 mutations in ihh and ks is currently unknown , but is probably low . by screening 100 patients , we could determine if the prevalence was at least 1% .
since kal1 mutations have been exclusively identified in anosmic / hyposmic patients , the majority of patients studied ( 40/54 ) were anosmic / hyposmic .
all patients were randomly selected from available dna samples without regard to prior mutation analysis from our large ihh / ks cohort .
dna was extracted from white blood cells in all ihh / ks using standard methods .
mlpa was performed in patients according to schouten ( schouten et al . , 2002 ;
a total of 125 ng of dna was hybridized overnight to the probe sets of the commercially available kits .
the p132 kal1 kit ( mrc holland , amsterdam , netherlands ) contains 34 probe pairs for all 14 kal1 exons and control sequences for other regions on xp , xq and yq11 .
the p133 kal2 kit contains 39 probe pairs covering the following exons of autosomal genes : gnrh1 ( exons 13 of 4 exons ) , gnrhr ( exons 13 of 3 exons ) , gpr54 ( exons 1 , 4 and 5 of 5 exons ) , fgfr1 ( 13 , 5 , 6 , 8,10,13,14 and 18 of 18 exons ) and nelf ( exons 5 , 11 and 15 of 16 exons )
. these probes also include control sequences on chromosomes 13 , 6 , 11 , 1517 , 19 , 20 and y. following hybridization , a ligation reaction was performed which served as a template for 35 cycles of multiplex pcr using universal primers .
pcr products were analyzed on an abi 310 autoanalyzer using the gene scan program and data were evaluated using genotyper 2.0 ( both from applied biosystems , foster city , ca , usa ) .
peak areas for each exon were converted into an excel file and the relative copy number of each fragment was compared to the same fragments from 2 to 3 controls .
deletions were confirmed by pcr and dna sequencing ( bhagavath et al . , 2007 ) .
one - hundred and twelve probands with ihh / ks were studied by mlpa ( table  i ) .
absent / impaired puberty by age 17 in girls and age 18 in boys , low serum gonadotrophins , no evidence of a pituitary tumor and otherwise normal pituitary function ( crowley et al . , 1985 ; bhagavath et al . ,
all males had total testosterone < 100 ng / dl ( normal 3001100 ng / dl ) , and all females had hypoestrogenic amenorrhea .
complete ihh was defined as absent breast development in females and testicular size 3 ml in males ( bhagavath et al . , 2006 ) .
incomplete ihh suggested the presence of prior steroid production and was defined as breast development of tanner 2 in females and testis size > 3 ml in males .
patient characteristics are shown in table  i. the medical college of georgia human assurance committee approved this study ; each patient signed informed consent .
fifty - four patients were studied for the presence of kal1 deletions ( kal1 kit ) and 100 ihh / ks patients were studied for deletions of an autosomal panel of fgfr1 , gnrh1 , gnrhr , gpr54 and nelf genes ( kal2 kit ) .
we estimated that we needed a sample size of 50 for study of kal1 gene deletions , since the prevalence of mutations is about 6% in anosmic males ( bhagavath et al . , 2007 ) .
the sample size for the autosomal gene panel ( kal2 kit ) was estimated to be 100 .
the prevalence of fgfr1 mutations in both normosmic ihh and ks is about 10% ( pitteloud et al . , 2006 ) , whereas gnrhr mutations occur in 34% of normosmic ihh ( bhagavath et al . , 2005 ) .
the prevalence of gnrh1 , nelf and gpr54 mutations in ihh and ks is currently unknown , but is probably low . by screening 100 patients , we could determine if the prevalence was at least 1% .
since kal1 mutations have been exclusively identified in anosmic / hyposmic patients , the majority of patients studied ( 40/54 ) were anosmic / hyposmic .
all patients were randomly selected from available dna samples without regard to prior mutation analysis from our large ihh / ks cohort .
dna was extracted from white blood cells in all ihh / ks using standard methods .
mlpa was performed in patients according to schouten ( schouten et al . , 2002 ;
a total of 125 ng of dna was hybridized overnight to the probe sets of the commercially available kits .
the p132 kal1 kit ( mrc holland , amsterdam , netherlands ) contains 34 probe pairs for all 14 kal1 exons and control sequences for other regions on xp , xq and yq11 . the p133 kal2 kit contains 39 probe pairs covering the following exons of autosomal genes : gnrh1 ( exons 13 of 4 exons ) , gnrhr ( exons 13 of 3 exons ) , gpr54 ( exons 1 , 4 and 5 of 5 exons ) , fgfr1 ( 13 , 5 , 6 , 8,10,13,14 and 18 of 18 exons ) and nelf ( exons 5 , 11 and 15 of 16 exons ) .
these probes also include control sequences on chromosomes 13 , 6 , 11 , 1517 , 19 , 20 and y. following hybridization , a ligation reaction was performed which served as a template for 35 cycles of multiplex pcr using universal primers .
pcr products were analyzed on an abi 310 autoanalyzer using the gene scan program and data were evaluated using genotyper 2.0 ( both from applied biosystems , foster city , ca , usa ) .
peak areas for each exon were converted into an excel file and the relative copy number of each fragment was compared to the same fragments from 2 to 3 controls .
deletions were confirmed by pcr and dna sequencing ( bhagavath et al . , 2007 ) .
using mlpa for the kal1 gene , deletions were identified in 4/54 ( 7.4% ) patients and in 4/40 ( 10% ) of anosmic / hyposmic patients ( table 
when only anosmic males were considered , kal1 deletions were present in 4/33 ( 12.1% ) .
three whole gene deletions were detected , as indicated by absence of peaks for each exon ( fig .  1 ) .
all three whole gene deletions were confirmed by the absence of bands by pcr . in one of these patients ,
deletion of exons 113 had previously been identified ( bhagavath et al . , 2007 ) ; however , dna sequencing of exon 14 was consistent with pseudogene sequence , thereby confirming the whole gene deletion by mlpa .
1 ) which , upon dna sequencing , revealed a 3 bp deletion ( tgt ) of codon 164 , deleting a cys ( cys164del ) rather than the deletion of the entire exon .
no heterozygous / homozygous deletions were identified in the fgfr1 , gnrh1 , gnrhr , gpr54 or nelf genes in 100 ihh / ks patients .
( a ) the kal1 exon 4 deletion ; ( b ) whole kal1 gene deletion ; ( c ) normal control .
arrows indicate the deleted exons . the horizontal axis ( top ) shows the size of the fragment in base pairs .
a difference in relative copy peak height or peak area indicates a copy number change of the probe target sequence .
the genetic basis of ihh / ks has been identified in 1520% of patients , most commonly in kal1 , gnrhr or fgfr1 genes ( bhagavath and layman , 2007 ) .
kal1 mutations have been identified in anosmic males  approximately 5% of ks males without a family history and 3070% of those with clear x - linked recessive inheritance ( bhagavath et al . , 2007 ) .
gnrhr mutations cause autosomal recessive ihh in 35% of ihh patients , all of whom are normosmic ( bhagavath et al . ,
interestingly , fgfr1 mutations occur in 10% of anosmic or normosmic patients ( pitteloud et al . ,
in contrast , only a very few gross deletions been identified in ihh / ks candidate genes .
available prevalence studies likely underestimate ihh / ks gene deletions as traditional pcr - based dna sequencing is unable to detect heterozygous gene deletions .
therefore , it is possible that a substantial number of mutations remain undiagnosed . using mlpa ,
heterozygous intragenic deletions have been found in 1520% of patients with cystic fibrosis ( audrezet et al . , 2004 ) ,
deafness ( del castillo et al . , 2003 ) and breast / ovarian cancer ( hogervorst et al . , 2003 ) who did not have point mutations .
mlpa is an effective technique used to detect genomic deletions and duplications ( schouten et al . , 2002 ) .
if probe pairs for individual exons are utilized , mlpa can successfully determine the relative copy number of all exons within gene / genes simultaneously .
mlpa has gained acceptance in genetic diagnostic laboratories due to its simplicity , relatively low cost and capacity for reasonably high throughput analysis .
( gerdes et al . , 2005 ) , analysis of dna methylation ( procter et al . , 2006 ) , relative mrna quantification ( wehner et al . ,
2005 ) , chromosomal characterization of cell lines and tissue samples ( wilting et al . , 2006 ) and the detection of polymorphisms ( volikos et al . , 2006 ) .
the frequency of gene deletions in ihh / ks candidate genes has not been previously reported .
we hypothesized that mlpa would identify intragenic deletions in 1520% of ihh / ks patients . using mlpa
three patients had whole gene deletions  one of which was previously described by pcr to be deleted of exons 113 ( bhagavath et al . , 2007 ) .
mlpa also revealed one subject who appeared to have a deletion of exon 4 . in this case ,
it is likely that the probe pair did not specifically anneal to the template of exon 4 , therefore , no ligation and subsequent pcr could be performed ( schouten et al . , 2002 ) .
our findings indicate that putative deletions indicate identified by mlpa requires confirmation by another technique .
probe pairs were dispersed across the coding regions of these genes , affording the opportunity to detect intragenic deletions .
only three exons were studied for the nelf gene , but at the time that this kit was designed , no nelf mutations had been described .
we can not exclude that intragenic deletions of exons not included in the kits occur , which would underestimate the prevalence of intragenic gene deletions .
nevertheless , no deletions of any of the five autosomal genes were identified in 100 ihh / ks patients . although the prevalence of deletions in ihh / ks in autosomal genes was much less than that expected , the prevalence of kal1 deletions approximated 1015% that we hypothesized .
the findings from this pilot study demonstrate the feasibility of mlpa to detect deletions in ihh / ks and suggest the benefits of future studies with an increased sample size of ihh / ks .
 idiopathic hypogonadotropic hypogonadism ( ihh ) and kallmann syndrome ( ks ) are clinically and genetically heterogeneous disorders caused by a deficiency of gonadotrophin - releasing hormone ( gnrh ) . 
 mutations in three genes  
 kal1 , gnrhr and fgfr1account for 1520% of all causes of ihh / ks . 
 nearly all mutations are point mutations identified by traditional pcr - based dna sequencing . the relatively new method of multiplex ligation - dependent probe amplification ( mlpa ) has been successful for detecting intragenic deletions in other genetic diseases . 
 we hypothesized that mlpa would detect intragenic deletions in 1520% of our cohort of ihh / ks patients . 
 fifty - four ihh / ks patients were studied for kal1 deletions and 100 were studied for an autosomal panel of fgfr1 , gnrh1 , gnrhr , gpr54 and nelf gene deletions . of all male and female subjects 
 screened , 4/54 ( 7.4% ) had kal1 deletions . 
 if only anosmic males were considered , 4/33 ( 12.1% ) had kal1 deletions . 
 no deletions were identified in any of the autosomal genes in 100 ihh / ks patients . 
 we believe this to be the first study to use mlpa to identify intragenic deletions in ihh / ks patients . 
 our results indicate 12% of ks males have kal1 deletions , but intragenic deletions of the fgfr1 , gnrh1 , gnrhr , gpr54 and nelf genes are uncommon in ihh / ks . 
Introduction
Materials and Methods
Patients
Molecular analysis/MLPA
Results
Discussion
Funding