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. 2017 May 1;102(5):1557-1567.
doi: 10.1210/jc.2016-3677.

Paternally Inherited DLK1 Deletion Associated With Familial Central Precocious Puberty

Andrew Dauber  1 , Marina Cunha-Silva  2 , Delanie B Macedo  2 , Vinicius N Brito  2 , Ana Paula Abreu  3 , Stephanie A Roberts  4 , Luciana R Montenegro  2 , Melissa Andrew  1 , Andrew Kirby  5 , Matthew T Weirauch  6 , Guillaume Labilloy  7 , Danielle S Bessa  2 , Rona S Carroll  3 , Dakota C Jacobs  8 , Patrick E Chappell  8 , Berenice B Mendonca  2 , David Haig  9 , Ursula B Kaiser  3 , Ana Claudia Latronico  2
Affiliations

Paternally Inherited DLK1 Deletion Associated With Familial Central Precocious Puberty

Andrew Dauber et al. J Clin Endocrinol Metab. .

Abstract

Context: Central precocious puberty (CPP) results from premature activation of the hypothalamic-pituitary-gonadal axis. Few genetic causes of CPP have been identified, with the most common being mutations in the paternally expressed imprinted gene MKRN3.

Objective: To identify the genetic etiology of CPP in a large multigenerational family.

Design: Linkage analysis followed by whole-genome sequencing was performed in a family with five female members with nonsyndromic CPP. Detailed phenotyping was performed at the time of initial diagnosis and long-term follow-up, and circulating levels of Delta-like 1 homolog (DLK1) were measured in affected individuals. Expression of DLK1 was measured in mouse hypothalamus and in kisspeptin-secreting neuronal cell lines in vitro.

Setting: Endocrine clinic of an academic medical center.

Patients: Patients with familial CPP were studied.

Results: A complex defect of DLK1 (∼14-kb deletion and 269-bp duplication) was identified in this family. This deletion included the 5' untranslated region and the first exon of DLK1, including the translational start site. Only family members who inherited the defect from their father have precocious puberty, consistent with the known imprinting of DLK1. The patients did not demonstrate additional features of the imprinted disorder Temple syndrome except for increased fat mass. Serum DLK1 levels were undetectable in all affected individuals. Dlk1 was expressed in mouse hypothalamus and in kisspeptin neuron-derived cell lines.

Conclusion: We identified a genomic defect in DLK1 associated with isolated familial CPP. MKRN3 and DLK1 are both paternally expressed imprinted genes. These findings suggest a role of genomic imprinting in regulating the timing of human puberty.

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Figures

Figure 1.
Figure 1.
Pedigree of family 1. Affected individuals are filled in black. All affected individuals carry the DLK1 mutation. Unaffected carriers, due to the imprinted pattern of inheritance, are marked with a black dot (II.2 and II.5). Individuals whose DNA was tested and did not carry the deletion are marked with the letters WT (wild-type). All wild-type individuals had normal pubertal timing. Individuals marked by empty symbols (I.2, II.6, II.7) were not tested. An “x” denotes that the subject underwent exome sequencing; a “g” denotes that the subject underwent whole-genome sequencing; and “s” denotes that the subject underwent genome-wide SNP microarray genotyping; and an “m” denotes that the subject underwent regional MLPA analysis.
Figure 2.
Figure 2.
Complex genomic rearrangement resulting in deletion of exon 1 of DLK1. (a) Screenshot of DLK1 region from the University of California Santa Cruz Genome Browser including two RefSeq DLK1 transcripts in dark blue. The region of the genomic deletion is highlighted in red, and the region of the duplication is highlighted in blue. The location of the PCR primers used to amplify the sequence of the deletion (c and d) are indicated by green arrows. (b) Screenshots from the Integrative Genomics Viewer of whole-genome data from subject III.2. In each panel, the top track lists the genomic coordinates shown on the screen. The middle section shows individual sequencing reads as gray bars with a histogram of individual base coverage above the reads. The colored reads represent individual paired-end sequence reads whose paired read is not located at the expected distance. This occurs due to the deletion/genomic rearrangement. Top left panel: overview of genomic region including DLK1 and its upstream region. The heterozygous deletion is clearly visible in the middle of the panel as a region of decreased sequencing coverage. The location of the DLK1 gene is noted at the bottom of this panel. Bottom left panel: zoomed-in view of the beginning of the deletion demonstrating a precipitous drop in sequencing coverage along with an abundance of reads with pairs at an unexpected distance. Top right panel: zoomed-in view of the end of the deletion. Bottom right panel: area of duplication in intron 3 of DLK1 showing increased coverage and abundance of reads with pairs at an unexpected distance. Blue arrows in all panels indicate the boundaries of the deletion or duplication. (c) PCR products demonstrating segregation of the deletion in the family. PCR primers span the deletion, and only individuals with the deletion demonstrate a PCR product of ∼850 bp. Subjects II.1, II.8, and III.5 were also tested and do not carry the deletion (data not shown). (d) Sanger sequencing of the PCR products in a subject with the complex genomic rearrangement. The sequence from intron 3 has been duplicated and inserted between the two ends of the large deletion. This results in a sequence that starts upstream of the deletion and then includes the whole duplicated region of intron 3 and concludes downstream of the deletion. The top panel’s sequence begins upstream of the deletion and shows the breakpoint where the upstream region joins the beginning of the duplicated region from intron 3 of DLK1. The bottom panel’s sequence starts with the end of the intron 3 duplicated region and joins to the region immediately downstream of the deletion in intron 1 of DLK1. All coordinates are given in the Genome Reference Consortium build 37.
Figure 3.
Figure 3.
DLK1 serum levels in controls and in five patients with CPP due to DLK1 deletion. Affected patients had undetectable DLK1 serum levels (below the assay limit of detection of 0.4 ng/mL).
Figure 4.
Figure 4.
Dlk1 mRNA expression in mouse MBH and in two kisspeptin-expressing mouse cell lines (KTaR-1 and KTaV-3). Dlk1 mRNA levels were measured by real-time quantitative PCR. HEK293 cells were used as a negative control. Results are shown as mean ± standard error of the mean for each sample (n = 3). Means with different letters are significantly different from one another (Tukey honest significant difference test, P < 0.05). Specifically, expression levels in HEK293 cells are significantly different than in KTaR-1 and KTaV-3 cells, which are also significantly different from MBH. There is no significant difference between KTaR-1 and KTaV-3. KTaR-1 is an arcuate-derived immortalized mouse cell line; KTaV-3 is an anteroventral periventricular-derived immortalized mouse cell line.
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