Mutations in two genes encoding different subunits of a receptor signaling complex result in an identical disease phenotype

Abstract

Polycystic lipomembranous osteodysplasia with sclerosing leukoencephalopathy (PLOSL), also known as "Nasu-Hakola disease," is a globally distributed recessively inherited disease leading to death during the 5th decade of life and is characterized by early-onset progressive dementia and bone cysts. Elsewhere, we have identified PLOSL mutations in TYROBP (DAP12), which codes for a membrane receptor component in natural-killer and myeloid cells, and also have identified genetic heterogeneity in PLOSL, with some patients carrying no mutations in TYROBP. Here we complete the molecular pathology of PLOSL by identifying TREM2 as the second PLOSL gene. TREM2 forms a receptor signaling complex with TYROBP and triggers activation of the immune responses in macrophages and dendritic cells. Patients with PLOSL have no defects in cell-mediated immunity, suggesting a remarkable capacity of the human immune system to compensate for the inactive TYROBP-mediated activation pathway. Our data imply that the TYROBP-mediated signaling pathway plays a significant role in human brain and bone tissue and provide an interesting example of how mutations in two different subunits of a multisubunit receptor complex result in an identical human disease phenotype.

Figure 1

Pedigrees, haplotypes, and TREM2 mutations in the Norwegian (A) and Swedish (B) families with PLOSL. Black symbols denote patients with PLOSL; half-black symbols denote heterozygous carriers of a mutated TREM2 allele; white symbols denote individuals who carry two wild-type alleles of TREM2. The 10-cM haplotypes were constructed by genotyping the following markers in both families: D6S1616-D6S1575-D6S1549 (from top to bottom in the figure). nt = position of mutated nucleotide. A, Haplotype 1-8-3 and 558G→A mutation, showing cosegregation with PLOSL in the Norwegian family. Haplotype 4-8-3 in the male carrier probably results from a recombination. B, Haplotype 4-7-3 and 233G→A substitution, cosegregating with PLOSL in the Swedish family.

Figure 2

Schematic presentation of the identified PLOSL mutations in TREM2. The positions of other loci for TYROBP-associated cell-surface receptors, TREM1 and LY95, as well as of the polymorphic “D” markers used in the segregation analysis, are indicated.

Figure 3

Northern-blot analysis of human tissues, with radiolabeled human TREM2 and TYROBP cDNAs used as probes. We hybridized human multiple-tissue northern blots (Clontech), with each lane containing 2 μg of poly(A)⁺ RNA, and a multiple-tissue mRNA expression array (Clontech) (J.P., unpublished data), using a [³²P]-labeled TREM2 cDNA probe, according to the manufacturer’s instructions. We generated the probe, corresponding to the transcribed region of TREM2, by PCR, from a cDNA clone (GenBank accession number BF343916) obtained from the IMAGE Consortium. We labeled and purified the probe by using the Rediprime II Random Prime Labelling System (Amersham Pharmacia Biotech) and the QIAquick Nucleotide Removal Kit (Qiagen) and performed the hybridizations by using ExpressHyb hybridization solution, according to the manufacturer’s (Clontech) protocol. A strong steady-state TYROBP mRNA signal is observed in hematological cells and tissues such as peripheral blood leukocytes (PBL) and spleen, whereas TREM2 can be detected only in lymph nodes. The intensities of the steady-state mRNA signals of TREM2 and TYROBP in different parts of the CNS are similar. In the CNS, relatively strong northern-blot signals can be detected in the basal ganglia (putamen and caudate nucleus), medulla, spinal cord, and corpus callosum. TYROBP northern-blot data have been published elsewhere (Paloneva et al. 2000).

Figure 4

Quantitative RT-PCR analysis of the expression of TREM2 (A) and TYROBP (B). Monocytes were stimulated with either a cytokine combination (comprising M-CSF, RANKL, and IL-1β) (cc) or pseudosynovial (PS) fluid, for 24 h, along the osteoclastic lineage. Human monocytes from healthy individuals were isolated from buffy-coat cells over Ficoll-Paque (Amersham Pharmacia Biotech). Mononuclear cells were collected, washed with PBS, and resuspended in serum-free macrophage medium (GIBCO) with antibiotics. Approximately 10–15×10⁶ cells/well were allowed to adhere to six-well plates, for 1 h at 37°C. Nonadherent cells were washed away with PBS, and mononuclear cells were stimulated, for 24 h, with either cytokines (i.e., M-CSF, RANKL, and IL-1β) or PS fluid. Stimulations were performed four times, in duplicate. Total RNA was isolated by use of TRIzol reagent, according to the manufacturer's instructions (GibcoBRL/Life Technologies). The RNA concentration was measured spectrophotometrically, and the quality was ascertained by ethidium bromide agarose gel. Three micrograms of the RNA was treated with DNase, and 2 μg of RNA was transcribed to cDNA (SuperScript Preamplification System; GibcoBRL). The number of copies of TYROBP and TREM2 in stimulated cells was determined by quantitative RT-PCR amplification from 200 ng of cDNA in LightCycler SYBR Green I PCR mix, by a LightCycler PCR machine (Roche/Molecular Biochemicals). The identity of the product was verified by melting-curve analysis. Serial dilutions of human TREM2 cDNA (GenBank accession number BF343916) and of TYROBP cDNA (GenBank accession number AA481924), cloned in a plasmid vector, were used to determine the copy number of the amplicon per 1,000 copies of ACTB (β-actin) cDNA (GenBank accession number X00351). Each individual sample was amplified at least twice for both genes. Statistical analyses were performed by one-way ANOVA with Bonferroni's multiple-comparison test (GraphPad Prism, version 3.00 for Windows; GraphPad Software). The values are expressed as the mean ± SEM of copies per 1,000 ACTB mRNA. Neg = negative controls. A, Expression of TREM2, which is increased in both the cc-stimulated (234 ± 195 copies of mRNA) and PS fluid–stimulated (174 ± 55 copies of mRNA) monocytes, compared with that in the negative controls (96 ± 58 copies of mRNA). However, the difference remained statistically nonsignificant (P>.05). B, Expression of TYROBP, which also is increased in cc-stimulated (27,190 ± 10,270 copies of mRNA; P>.05) and PS fluid–stimulated (54,740 ± 18,220 copies of mRNA; P<.05, denoted by an asterisk [*]) cells, compared with that in the negative controls (14,210 ± 6,290 copies of mRNA).