Association of hereditary hemorrhagic telangiectasia and hereditary nonpolyposis colorectal cancer in the same kindred



Endoglin (CD105) is a proliferation-associated protein that is strongly expressed in endothelial tissue and has a role in tumor angiogenesis. Mutations in endoglin are also linked to Hereditary Hemorrhagic Telangiectasia type 1 (HHT1), an autosomal dominant disease associated with aberrant angiogenesis. We report an unusual association of HHT1 and Hereditary Nonpolyposis Colorectal Cancer (HNPCC) in the same kindred. Genetic analysis indicates that these 2 syndromes are genetically unrelated and separately segregated within the family. The mutation in the endoglin gene leads to a truncated protein. The mutation in the mismatch repair gene MLH1 causes a splicing defect, giving synthesis to an unstable mRNA from this mutated allele. The potential protective role of an endoglin mutation in patients with HNPCC is discussed. © 2005 Wiley-Liss, Inc.

Endoglin (CD105), a proliferation-associated glycoprotein, is a component of the transforming growth factor-β (TGF- β) signal transduction pathway.1 Endoglin is known to bind to TGF-β1 and TGF-β3 and interacts with TGF-β receptor I and II. The biological action of endoglin is to modulate downstream phosphorylation of specific SMAD proteins that mediate the effects of TGF-β.2 Endoglin has been shown to promote angiogenesis and neovascularization, and is strongly expressed in blood vessels of tumor tissue.3 CD105 is often used as a method of measuring tumor microvessel density, and its levels have prognostic significance in multiple tumor types.

Both hereditary hemorrhagic telangiectasia type 1, (HHT1) and hereditary nonpolyposis colorectal cancer (HNPCC) are associated with abnormal expression of endoglin. HHT is an autosomal dominant disease characterized by vascular malformations (e.g., telangiectasias and arteriovenous malformations of skin, mucosa and viscera).4 The diagnosis of HHT is based on the criteria developed by Plauchu et al.5, 6 HHT is caused by mutations in either the endoglin (HHT1, chromosome 9q33–34) or ALK1 (HHT2, chromosome 12q13), both of which are components of the transforming growth factor-β (TGF- β) signal transduction pathway.1 In HHT1, levels of normal endoglin are diminished resulting in aberrant blood vessel development. Studies in endoglin knockout-mice indicate that many of the morphologic abnormalities of HHT1 are a result of abnormal TGF signaling.7

Hereditary Nonpolyposis Colorectal Cancer (HNPCC) is a disorder that is often associated with abnormally high levels of endoglin and abnormal TGF-β signaling.8, 9 This disorder is inherited in an autosomal-dominant manner and diagnosis is based on the Amsterdam criteria.10, 11 In most families who fulfill the Amsterdam criteria, germline mutations in the DNA mismatch repair (MMR) genes have been identified. Microsatellite DNA sequences are susceptible to mutation formation, and damaged MMR function results in microsatellite instability (MSI).10, 12MLH1 (chromosome 3p21) and MSH2 (chromosome 2p16) account for almost 90% of all the identified HNPCC mutations.13, 14, 15 Kindreds sharing the same mutation in the DNA MMR genes may exhibit significant phenotypic variation such as the age of onset.16 This variation has often been related to modifier genes, and increased frameshift mutations of tandem repeats found in the coding region of specific genes.17 One such affected gene is the TGF-β IIR, which is often mutated in both HNPCC and sporadic colorectal cancer and may be associated with increased carcinogenesis due to altered TGF-β signal transduction.17, 18

In our report, we discuss a kindred with both Hereditary Hemorrhagic Telangiectasia type 1 (HHT1) and Hereditary Nonpolyposis Colorectal Cancer (HNPCC). The presence of both HHT1 and HNPCC in the same individual raises the possibility that lower endoglin levels might contribute to a delayed growth of solid tumors in HNPCC patients, making them relatively resistant to metastatic cancer.


HHT, hereditary hemorrhagic telangiectasia; HNPCC, hereditary nonpolyposis colorectal cancer; TGF-β, transforming growth factor-β; MMR, mismatch repair.


Subjects and clinical material

The family members studied are of Jewish Moroccan origin. A description of the family pedigree, manner of inheritance and clinical features were obtained by detailed questioning and examination of available family members.

DNA and cDNA preparation

After receiving informed consent, venous blood samples from the proband and first and second-degree family members were obtained. Genomic DNA was extracted using Puregene DNA isolation kit (Gentra system, Minneapolis, MN, catalog number D-k50). DNA extraction from paraffin embedded tissue was performed using QIAamp DNA Mini Kit, following the Tissue Protocol (Qiagen, Chatsworth, CA; catalog number 51304). Total RNA was isolated from lymphocytes using Tri- Reagent solution (MRC, Cincinnati, OH, catalog number TR118) and cDNA was transcribed from 1 μg of RNA using a random hexamer mixture as primers and M-MLV reverse transcriptase (Promega, Madison, WI).

Linkage analysis

Primers for PCR amplification of microsatellite markers were derived from the genomic sequence of ALK1 (Genebank accession number D12S325) and ENG (Genebank accession number D9S904, D9S918 and D9S1821). PCR products were analyzed by electrophoresis on 8 M urea, 8% polyacrylamide gels (500 V, 20 hr), and were visualized by silver staining.

DNA sequencing

Exonic sequences of the endoglin and the MLH1 genes were amplified using primers from the flanking introns sequences.14, 19 PCR products were subjected to direct sequencing.

ENG-mutation screening

Exon 4 of the endoglin gene was amplified with primers derived from the flanking sequence of the mutation, within the exon. (F: 5′- CTGAGCTGAATGACCCCCAGAGCATCCTCCT-3′; R: 5′-GGCTTGTGGCATGTGAACTGTGGCACAGCGTG-3′). PCR products (182 bp) were subjected to direct sequencing.

MLH1-mutation screening

Exon 10 of the MLH1 gene was amplified with primers derived from the flanking sequence of the mutation within the exon. F: 5′-TGCCCAAAAACACACACCCATTCCTGTACCCC-3′ (underlined C is substituted for T to create a Sma1 restriction sites within the sequence of the affected DNA samples); R: 5′-CTGTGCCTTGTACCTGTAAGAAGGGACAGAAC-3′ (see Fig. 2). Restriction analysis of PCR products with Sma1 enzyme of all family members was performed in order to detect mutation carriers.

Figure 2.

Segregation of HNPCC within the family. (a) Wild-type and mutant sequences of MLH1 genes are described. Forward primer contains a point mutation (T > C) compared to wild-type sequence creating a Sma 1 recognition site in the mutated allele. (b) Pedigree of HHT1 and HNPCC family. Gel electrophoresis of PCR product after digestion with Sma 1. The mutant allele is cleaved to create a 167 bp fragment. The wild-type allele is resistant to digestion (197 bp).Black symbols = HHT1; Circled symbols = HNPCC; numbers = Age of HNPCC patients when diagnosed.

Real-time PCR reaction

Real-time quantitative PCR analysis was performed with an automated sequence detection system (ABI Prism 7000, Applied Biosystems, Weiterstadt, Germany). The PCR reaction mix (20 μl) was composed of 10 μl Syber Universal PCR Master Mix (ABI, Warrington UK), 1 μl cDNA (each sample in a quadruplicate) and a final concentration of 500 nM from each primer. The amplified fragment of the MLH1 cDNA contains the junction sequence between exon 10 and exon 11. L19 cDNA was used as an internal standard. The following primers were used: F (MLH1exon10) 5′-AACAGTGTATGCAGCCTATTTG-3′; R (MLH1exon11) 5′-CACATTCTGGGGACTGATTTC-3′; F (L19exon2): 5′-CCCAATGAGACCAATGAAATC-3′; R (L19exon3): 5′:-ATGGACCGTCACAGGCTTG-3′.

MLH1 mRNA quantification

For each reaction tube, the fluorescence signal of the reporter dye was divided by the fluorescence signal of the passive reference dye to obtain a ratio defined as the normalized reporter signal. The threshold line was set at a ratio of 0.02.20 The point at which the amplification plot crossed this threshold was defined as Ct, which represented the cycle number at this point. Final results at each data point are the average of a quadruplicates (i.e., each cDNA was analyzed in a quadruplicate to confirm equal volume loading). MLH1 mRNA levels were obtained by subtracting the average Ct of the control reaction (L19) from that of the average MLH1 reaction using the calculation 2−(Δct), as previously described.20


Family description

A description of the family pedigree, manner of inheritance and clinical features are presented in Figure 1. The proband is a 21-year-old woman (Fig. 1, 3rd generation) who presented with hemoglobin level of 19 g/dl on a routine blood test. She was mildly cyanotic, had finger clubbing and several telangiectases on her mucous membranes and thumbnails. Blood tests showed hypoxia without acidosis. Chest CT revealed a large arteriovenous malformation in the left upper lobe and a small one on the right middle lobe. The patient also had a positive family history of epistaxis and telagiectasias. Three generations of this kindred were evaluated, and 11 clinically affected individuals were identified (Fig. 1). In the first generation, 1 male individual suffered from severe epistaxis and anemia requiring episodic blood transfusions. He died in his sixth decade from anaphylaxis related to a blood transfusion. A second female, age 78 years, has intermittent epistaxis and several telangiectases on her tongue, lips and thumbnails. She had colon cancer at the age of 60 and underwent hemicolectomy with no recurrence to date. Of 6 affected subjects in the second generation, 5 have intermittent epistaxis and all have multiple telangiectases located on their lips and oral cavity. None had gastrointestinal bleeding, and there was no evidence of visceral arteriovenous malformation in any of the individuals. In the second generation, a 56-year-old woman had colon cancer at the age of 52 years, which was treated with local resection and adjuvant chemotherapy with no recurrence to date (Fig. 2). Her brother died at the age of 41 years of disseminated colon cancer. In the third generation, apart from the proband, all have intermittent epistaxis, but without associated anemia. The proband's sister, at the age of 24 years, has a small, clinically nonsignificant, pulmonary arteriovenous malformation found by chest computerized tomography.

Figure 1.

Pedigree of a HHT family demonstrating linkage to the endoglin gene. DNA was extracted from family members and haplotype analysis was performed with 3 polymorphic markers (D9S904, D9S918 and D9S1821) flanking the endoglin locus. Haplotype 232 segregates with clinical manifestations of HHT. Arrow = proband; Black symbols = HHT1; Circled symbols = HNPCC.

HHT linkage analysis

Linkage analysis with the ALK1 associated polymorphic marker (D12S325) ruled out any involvement of ALK1 as a cause of HHT in this kindred. The endoglin gene is located within the interval D9S904-D9S1821 of 0.9 cM. Linkage analysis of the polymorphic markers associated with the endoglin gene (D9S904, D9S918 and D9S1821) indicates a segregation of the haplotype 232 with the clinical symptoms (Fig. 1, black symbols).

Detection of a mutation in exon 4 of the endoglin gene

Sequence analysis of the proband's endoglin gene revealed a nonsense mutation within exon 4. Amino acid arginine 171 (CGA) is mutated to a stop codon (TGA). DNA segments encompassing exon 4 from all family members were amplified and sequenced. The C511T mutation was detected in all DNA samples of family members that exhibited allele haplotype 232 and had clinical symptoms of HHT (Fig. 1).

Detection of a mutation in exon 10 of the MLH1 gene

Three family members, a mother and 2 children, developed colorectal tumors raising the possibility of HNPCC within this kindred (Fig. 1). Sequencing of all exons of the MLH1 gene revealed an A883G mutation. within the junctional sequence between exons 10 and 11.

MLH1-mutation screening

Exon 10 of all family members was amplified using primers as described in Material and Methods. The A883G mutation in the MLH1 gene creates a new recognition site for the restriction enzyme Sma1 (CCCGGG; Fig. 2). Screening of all family members by restriction enzyme analysis revealed no other carriers of this mutation except the 3 patients already diagnosed. No carriers were found among DNA samples of 70 unrelated Jewish Moroccan (data not shown), indicating that the A883G substitution represents a real mutation rather than a polymorphic site in this population.

The MLH1 mutation disrupts the processing of the mutated allele

The mutated nucleotide is the 2nd base upstream to the junction between exons 10 and 11. In order to determine if the mutation interferes with the processing of the MLH1 transcript, RNA was extracted from blood samples of affected and nonaffected subjects. Reverse-Transcriptase PCR amplification products of the junction sequence between exons 10 and 11 were subjected to direct sequencing. Sequence analysis of the amplified product deduced from the affected subject revealed only the wild-type sequence. Absence of the mutated transcript suggests early degradation of mRNA containing the mutation, probably because the mutated allele was not processed correctly. Real Time PCR (RTPCR) analysis verified this conclusion. Amplification of the junction area with Syber Green master mix, using L19 cDNA as a standard showed that the concentration of the RTPCR product of MLH1 of the affected subject is 0.5× the MLH1 mRNA concentration of the unaffected subject (Fig. 3).

Figure 3.

Effect of the mutation in MLH1 gene on gene expression. (a) DNA sequence of the junction area between exons 10/11 (thin vertical arrow) is presented, demonstrating the heterozygous mutation (A883G; thick vertical arrow) identified in the family. The mutation occurs in the second nucleotide upstream to the junction point in exon 10. RNA was extracted from blood of affected and control subjects and cDNA synthesis was performed by reverse transcriptase PCR. CDNA sequence of the affected individual exhibits the wild-type allele only, indicating that RNA product of the mutant allele is unstable, probably due to splicing defect. (b) RNA was extracted from blood of affected and control subjects and cDNA synthesis was performed by reverse transcriptase PCR. Quantification of cDNA levels of MLH1 and L-19 (internal standard) were performed by real time PCR. Amplification reactions were performed in triplicates. Relative cDNA levels are presented.


In our study, we describe a family with clinical features of both HHT1 and HNPCC that segregate independently within the kindred. Mutational analysis indicates that the gene responsible for the HHT1 phenotype is endoglin (CD105), and the C511T substitution generates a premature stop codon in exon 4 of the HHT1 gene. This previously reported mutation results in HHT1 clinical symptoms due to endoglin haploinsufficiency.21

Members of the family with manifestations of HNPCC had a mutation in the MLH1 gene located at the junction between exon 10 and 11, which resulted in a splicing defect leading to transcript instability and a reduction in the protein product. The consensus sequence at the 5′ end of the exon-intron junction site is AGGU.22 However, only the 2 nucleotides within the intron (GU) are invariable. Nevertheless, the transformation of A to G in the consensus sequence within exon 10 seems to abrogate the RNA maturation process of the mutated allele. The same mutation has been previously reported and associated with affected HNPCC individuals living in the Netherlands (, Wijnen et al., unpublished). Given that the A883G mutation is neither a normal variant of the general population nor of Moroccan Jews together with the fact that in 2 ethnically unrelated populations, the mutation was associated with manifestations of HNPCC would indicate that this substitution is not a benign polymorphism.

MLH1 (3p21) and MSH2 (2p16), 2 DNA mismatch repair genes, account for almost 90% of all the identified HNPCC mutations. Inactivation of the second copy of the MMR gene leads to high level of microsatellite instability and promotes mutagenesis at these sites. Tumorogenesis is believed to be a result of microsatellite instability in coding regions of specific genes.10

The association of HHT and HNPCC may be coincidental without any relationship between the genes and the clinical manifestations of these 2 diseases. The estimated population prevalence of HHT in Europe, USA and Japan is 1:5,000 to 1:8,000. The prevalence of colon cancer in European population is 176:100,00023 and 3% of these cases are HNPCC.24 The chance occurrence of both HHT and HNPCC is about 1:100,000,000.

An intriguing possibility is that there is a link between the 2 genes both at the genetic and clinical levels. In other forms of hereditary colorectal cancer, there is an association between HHT and genes responsible for tumorigenesis. There are several reports of families with both juvenile polyposis and manifestations consistent with HHT.25, 26, 27 Recently, Gallione et al.27 reported that mutations in SMAD4 (also known as MADH4), a protein involved in effecting downstream signaling of TGF-β, leads to a combined syndrome of juvenile polyposis and HHT. These authors postulate that SMAD 4, which is a common downstream effector of both endoglin (HHT) and BMPR1A (juvenile polyposis), mediated signaling may cause both HHT and juvenile polyposis when mutated as this protein is expressed in both endothelial and colonic mesenchymal cells.

Tumorigenesis in HNPCC can also involve the same signaling pathway as endoglin. In nearly all HNPCC tumors, the TGf-β IIR gene is often inactivated as a result of coding region microsatellite instability.18, 28, 29, 30 A small proportion of HNPCC tumors also have SMAD4 mutations. It has been postulated that the TGF-β pathway in many cell types has a tumor suppressor role7 and mutations of TGf-β IIR or SMAD4 are associated with transformation and metastatic potential.2, 31 At the same time, TGF-β may promote tumor angiogenesis via the endoglin pathway as knockouts of both TGF-β receptor 1 and 2 result in decreased vasculogenesis and abnormal blood vessels.7 How might endoglin mutations in this patient affect the defect in TGF-β signaling found in HNPCC? Endoglin has been a key element in angiogenesis, and high levels of endoglin are found in advanced colorectal tumors.32 Based on evidence from endoglin knockout mice strains (with different genetic backgrounds) or anti-sense inhibition of endoglin expression, it appears that endoglin is necessary for both recruitment of smooth muscle cells to developing endothelial tubes and the development of the complex structure necessary for stable blood vessels.2, 31, 33, 34 It would be expected then that the mutations in endoglin found in this family might have a protective effect. Families with germline mutations in the MLH1 have colorectal cancer diagnosed at an younger age (below 50 years).10 In our family, an “attenuated” HNPCC phenotype characterized by the later-onset of colorectal cancer onset in the 2 family members carrying both the endoglin and MLH1 mutations was observed. However, it should be noted that there is insufficient evidence within the reported family to make any conclusions regarding as to whether there is an improved outcome and delayed onset of colorectal cancer in members who have both mutations.

In summary, we have described a family in which there are both mutations of the endoglin and MLH1 genes, resulting in some family members having both HHT1 and HNPCC. It will be interesting to follow families with HHT1 to determine whether mutated endoglin has a mitigating effect on the manifestations of solid tumors.