Germline allele-specific expression of DAPK1 in chronic lymphocytic leukemia.

We previously reported a rare germline variant (c.1-6531) that resulted in allele-specific expression (ASE) of death-associated protein kinase 1 (DAPK1) and predisposition to chronic lymphocytic leukemia (CLL). We investigated a cohort of CLL patients lacking this mutation for the presence of ASE of DAPK1. We developed a novel strategy that combines single-nucleotide primer extension (SNuPE) with MALDI-TOF mass spectrometry, and detected germline DAPK1 ASE in 17 out of 120 (14.2%) CLL patients associated with a trend towards younger age at diagnosis. ASE was absent in 63 healthy controls. Germline cells of CLL patients with ASE showed increased levels of DNA methylation in the promoter region, however, neither genetic nor further epigenetic aberrations could be identified in the DAPK1 5' upstream regulatory region, within distinct exons or in the 3'-UTR. We identified B-lymphoid malignancy related cell line models harboring allelic imbalance and found that allele-specific methylation in DAPK1 is associated with ASE. Our data indicate that ASE at the DAPK1 gene locus is a recurrent event, mediated by epigenetic mechanisms and potentially predisposing to CLL.

Hereditary intrinsic factor deficiency in chaldeans.

Juvenile vitamin B(12) or cobalamin (Cbl) deficiency is notoriously difficult to explain due to numerous acquired and inherited causes. The consequences of insufficient Cbl are megaloblastic anemia, nutrient malabsorption, and neurological problems. The treatment is straightforward with parenteral Cbl supplementation that resolves most health issues without an urgent need to clarify their cause. Aside from being clinically unsatisfying, failing to elucidate the basis of Cbl deficiency means important information regarding recurrence risk is not available to the individual if the cause is contagious or inherited. Acquired causes have largely disappeared in the Modern World because they were mostly due to parasites or malnutrition. Today, perhaps the most common causes of juvenile Cbl deficiency are Imerslund-Gräsbeck syndrome and inherited intrinsic factor deficiency (IFD). Three genes are involved and genetic testing is complicated and not widely available. We used self-identified ancestry to accelerate and confirm the genetic diagnosis of IFD in three families of Chaldean origin. A founder mutation limited to Chaldeans from Iraq in the intrinsic factor gene GIF was identified as the cause. World events reshape the genetic structure of populations and inherited diseases in many ways. In this case, all the patients were diagnosed in the USA among recent immigrants from a single region. While IFD itself is not restricted to one kind of people, certain mutations are limited in their range but migrations relocate them along with their host population. As a result, self-identified ancestry as a stratifying characteristic should perhaps be considered in diagnostic strategies for rare genetic disorders.

Inherited cobalamin malabsorption. Mutations in three genes reveal functional and ethnic patterns.

BACKGROUND: Inherited malabsorption of cobalamin (Cbl) causes hematological and neurological abnormalities that can be fatal. Three genes have been implicated in Cbl malabsorption; yet, only about 10% of ~400-500 reported cases have been molecularly studied to date. Recessive mutations in CUBN or AMN cause Imerslund-Gräsbeck Syndrome (IGS), while recessive mutations in GIF cause Intrinsic Factor Deficiency (IFD). IGS and IFD differ in that IGS usually presents with proteinuria, which is not observed in IFD. The genetic heterogeneity and numerous differential diagnoses make clinical assessment difficult. METHODS: We present a large genetic screening study of 154 families or patients with suspected hereditary Cbl malabsorption. Patients and their families have been accrued over a period spanning >12 years. Systematic genetic testing of the three genes CUBN, AMN, and GIF was accomplished using a combination of single strand conformation polymorphism and DNA and RNA sequencing. In addition, six genes that were contenders for a role in inherited Cbl malabsorption were studied in a subset of these patients. RESULTS: Our results revealed population-specific mutations, mutational hotspots, and functionally distinct regions in the three causal genes. We identified mutations in 126/154 unrelated cases (82%). Fifty-three of 126 cases (42%) were mutated in CUBN, 45/126 (36%) were mutated in AMN, and 28/126 (22%) had mutations in GIF. We found 26 undescribed mutations in CUBN, 19 in AMN, and 7 in GIF for a total of 52 novel defects described herein. We excluded six other candidate genes as culprits and concluded that additional genes might be involved. CONCLUSIONS: Cbl malabsorption is found worldwide and genetically complex. However, our results indicate that population-specific founder mutations are quite common. Consequently, targeted genetic testing has become feasible if ethnic ancestry is considered. These results will facilitate clinical and molecular genetic testing of Cbl malabsorption. Early diagnosis improves the lifelong care required by these patients and prevents potential neurological long-term complications. This study provides the first comprehensive overview of the genetics that underlies the inherited Cbl malabsorption phenotype.

Heritable polymorphism predisposes to high BAALC expression in acute myeloid leukemia.

Overexpression of the brain and acute leukemia, cytoplasmic (BAALC) gene is implicated in myeloid leukemogenesis and associated with poor outcome in both acute myeloid leukemia (AML) and acute lymphoblastic leukemia patients. Additionally, high BAALC expression occurs in glioblastoma, melanoma, and childhood gastrointestinal stroma tumors, suggesting an oncogenic role for BAALC. However, the mechanisms underlying the deregulated expression are unknown. We hypothesized that a common heritable genetic feature located in cis might account for overexpression of BAALC in an allele-specific manner. By sequencing the genomic region of BAALC we identified nine informative single nucleotide polymorphisms (SNPs) and tested them for a possible association with BAALC expression levels. We show that BAALC overexpression occurs in the presence of the T allele of SNP rs62527607[GT], which creates a binding site for the activating RUNX1 transcription factor in the BAALC promoter region. The mechanism is demonstrated experimentally in vitro using luciferase reporter assays and electrophoretic mobility shift assay (EMSA) analysis. The association of high BAALC expression with the T allele and its correlations with RUNX1 expresser status are shown in vivo in a test set (n = 253) and validation set (n = 105) of samples from cytogenetically normal AML patients from different populations. Thus, we identify a heritable genomic feature predisposing to overexpression of an oncogene, thereby possibly leading to enhanced AML leukemogenesis. Our findings further suggest that genomic variants might become useful tools in the practice of personalized medicine.

miR-3151 interplays with its host gene BAALC and independently affects outcome of patients with cytogenetically normal acute myeloid leukemia.

High BAALC expression levels are associated with poor outcome in cytogenetically normal acute myeloid leukemia (CN-AML) patients. Recently, miR-3151 was discovered in intron 1 of BAALC. To evaluate the prognostic significance of miR-3151 expression levels and to gain insight into the biologic and prognostic interplay between miR-3151 and its host, miR-3151 and BAALC expression were measured in pretreatment blood of 179 CN-AML patients. Gene-expression profiling and miRNA-expression profiling were performed using microarrays. High miR-3151 expression was associated with shorter disease-free and overall survival, whereas high BAALC expression predicted failure of complete remission and shorter overall survival. Patients exhibiting high expression of both miR-3151 and BAALC had worse outcome than patients expressing low levels of either gene or both genes. In gene-expression profiling, high miR-3151 expressers showed down-regulation of genes involved in transcriptional regulation, posttranslational modification, and cancer pathways. Two genes, FBXL20 and USP40, were validated as direct miR-3151 targets. The results of the present study show that high expression of miR-3151 is an independent prognosticator for poor outcome in CN-AML and affects different outcome end points than its host gene, BAALC. The combination of both markers identified a patient subset with the poorest outcome. This interplay between an intronic miR and its host may have important biologic implications.

Juvenile cobalamin deficiency in a 17-year-old child with autonomic dysfunction and skin changes.

We report a rare case of juvenile cobalamin deficiency who presented at the age of 17 years. He was underweight and had skin changes, normocytic anemia, and autonomic dysfunction, which led to adynamic ileus and acute postrenal failure. The expected macrocytosis was masked by an underlying alpha-thalassemia trait. The patient had an excellent response to parenteral cobalamin treatment.

Familial peripheral keratopathy without PAX6 mutation.

PURPOSE: To describe the clinical features of a familial abnormality of the corneal stem cells and to investigate the role of PAX6 mutations in the affected family members. METHODS: A family with multiple generations of peripheral keratopathy was evaluated. Because of the corneal phenotypic similarity to aniridia-related keratopathy, it was hypothesized that the affected patients might have a dominantly inherited mutation of PAX6 on chromosome 11. Commercial sequencing of germline DNA from 1 affected family member did not identify any PAX6 mutations in the exons or intron-exon boundary regions. Because the commercial analysis is not designed to identify PAX6 deletions, germline DNA was collected from 5 unaffected and 2 additional affected family members. DNA repeat markers in the region of PAX6 were analyzed to determine whether this chromosomal region segregates with the disease phenotype. RESULTS: Affected family members with this autosomal dominant peripheral corneal abnormality showed evidence of progressive corneal stem cell dysfunction. Several individuals demonstrated corectopia, and 1 individual had ectropion uvea but no other iris or ocular abnormalities associated with aniridia. Genotyping data of affected and unaffected family members demonstrated that the PAX6 region does not segregate with the disease phenotype. CONCLUSIONS: The features of this autosomal dominant abnormality show some similarity to aniridia, although the classic characteristics of severe iris hypoplasia and macular hypoplasia are absent. Mutational screening and genotyping could not conclusively clarify a role for PAX6 in this disease phenotype, suggesting that it is a distinct clinical and genetic disease entity, not a variant of aniridia.

Ancient founder mutation is responsible for Imerslund-Gräsbeck Syndrome among diverse ethnicities.

BACKGROUND: Imerslund-Gräsbeck syndrome (IGS) was described just over 50 years ago by Olga Imerslund and Ralph Gräsbeck and colleagues. IGS is caused by specific malabsorption of cobalamin (Cbl) due to bi-allelic mutations in either the cubilin gene (CUBN) or the human amnionless homolog (AMN). Mutations in the two genes are commonly seen in founder populations or in societies with a high degree of consanguineous marriages. One particular mutation in AMN, c.208-2A>G, causing an out-of-frame loss of exon 4 in the mRNA, is responsible for some 15% of IGS cases globally. We present evidence that this founder mutation causes a substantial percentage of cases among diverse ethnicities and that the mutation is as old as human civilization. METHODS: Partial genotyping indicated a founder event but its presence in diverse peoples of Arabic, Turkish, Jewish, and Hispanic ancestry suggested that the mutation might be recurrent. We therefore studied the flanking sequence spanning 3.5 Mb to elucidate the origin of the haplotype and estimate the age of the mutation using a Bayesian inference method based on observed linkage disequilibrium. RESULTS: The mutation's distribution, the size of the shared haplotype, and estimates of growth rate and carrier frequency indicated that the mutation was a single prehistoric event. Dating back to the ancient Middle East around 11,600 BC, the mutation predates the advent of writing, farming, and the monotheistic religions of the region. CONCLUSIONS: This mutation causes over 50% of the IGS cases among Arabic, Turkish, and Sephardic Jewish families, making it a primary target for genetic screening among diverse IGS cases originating from the Middle East. Thus, rare founder mutations may cause a substantial number of cases, even among diverse ethnicities not usually thought to be related.

Variants in the netrin-1 receptor UNC5C prevent apoptosis and increase risk of familial colorectal cancer.

BACKGROUND & AIMS: Expression of the netrin-1 dependence receptor UNC5C is reduced in many colorectal tumors; mice with the UNC5C mutations have increased progression of intestinal tumors. We investigated whether specific variants in UNC5C increase risk of colorectal cancer (CRC). METHODS: We analyzed the sequence of UNC5C in blood samples from 1801 patients with CRC and 4152 controls from 3 cohorts (France, United States, and Finland). Almost all cases from France and the United States had familial CRC; of the Finnish cases, 92 of 984 were familial. We analyzed whether CRC segregates with the UNC5C variant A628K in 3 families with histories of CRC. We also performed haplotype analysis to determine the origin of this variant. RESULTS: Of 817 patients with familial CRC, 14 had 1 of 4 different, unreported missense variants in UNC5C. The variants p.Asp353Asn (encodes D353N), p.Arg603Cys (encodes R603C), and p.Gln630Glu (encodes Q630E) did not occur significantly more often in cases than controls. The variant p.Ala628Lys (A628K) was detected in 3 families in the French cohort (odds ratio, 8.8; Wald's 95% confidence interval, 1.47-52.93; P = .03) and in 2 families in the US cohort (odds ratio, 1.9; P = .6) but was not detected in the Finnish cohort; UNC5C A628K segregated with CRC in families. Three families with A628K had a 109-kilobase identical haplotype that spanned most of UNC5C, indicating recent origin of this variant in white subjects (14 generations; 95% confidence interval, 6-36 generations). Transfection of HEK293T cells with UNC5C-A628K significantly reduced apoptosis compared with wild-type UNC5C, measured in an assay of active caspase-3. CONCLUSIONS: Inherited mutations in UNC5C prevent apoptosis and increase risk of CRC.

[Homozygous mutation in the intrinsic factor gene in a child with severe vitamin B12 deficiency]. / Homozygot mutation i intrinsic factor-genet hos et barn med svær vitamin B 12-mangel.

A 28 month-old boy was hospitalized with pallor and weight stagnation. He had macrocytic anaemia and pancytopenia due to cobalamin deficiency and a rare homozygous mutation in the intrinsic factor gene. His sister showed similar symptoms at the age of 15 months. The heterozygous father had no symptoms, but did have a low cobalamin level. Gastroscopy with biopsies showed no pathology. All were given monthly cyanocobalamin injections which, however, caused leg cramps. Replacement with monthly hydroxocobalamin was successful.

Juvenile selective vitamin B12 malabsorption: 50 years after its description-10 years of genetic testing.

Fifty years have passed since the description of juvenile selective malabsorption of cobalamin (Cbl). Quality of life improvements have dramatically reduced the incidence of parasite-induced or nutritional Cbl deficiency. Consequently, inherited defects have become a leading cause of Cbl deficiency in children, which is not always expressed as anemia. Unfortunately, the gold standard for clinical diagnosis, the Schilling test, has increasingly become unavailable, and replacement tests are only in their infancy. Genetic testing is complicated by genetic heterogeneity and differential diagnosis. This review documents the history, research, and advances in genetics that have elucidated the causes of juvenile Cbl malabsorption. Genetic research has unearthed many cases in the past decade, mostly in Europe and North America, often among immigrants from the Middle East or North Africa. Lack of suitable clinical testing potentially leaves many patients inadequately diagnosed. The consequences of suboptimal Cbl levels for neurological development are well documented. By raising awareness, we wish to push for fast track development of better clinical tools and suitable genetic testing. Clinical awareness must include attention to ethnicity, a sensitive topic but effective for fast diagnosis. The treatment with monthly parenteral Cbl for life offers a simple and cost-effective solution once proper diagnosis is made.

Allele-specific expression of TGFBR1 in colon cancer patients.

The genetic component of colorectal cancer (CRC) predisposition has been only partially explained. We recently suggested that a subtle decrease in the expression of one allele of the TGFBR1 gene was a heritable quantitative trait predisposing to CRC. Here, we refined the measurements of allele-specific expression (ASE) of TGFBR1 in a population-based series of CRC patients and controls. Five single-nucleotide polymorphisms (SNPs) in the 3'-untranslated region of the gene were genotyped and used for ASE determination by pyrosequencing. After eliminating non-informative samples and samples with RNA of insufficient quality 109 cases and 125 controls were studied. Allelic ratios ranged between 0.74 and 1.69 without evidence of bimodality or cutoff points for 'ASE' versus 'non-ASE'. Treating ASE as a continuous variable, cases had non-significantly different values than controls (P = 0.081 when comparing means by permutation test). However, cases had significantly higher ASE values when comparing medians by permutation test (P = 0.0027) and when using Wilcoxon test (P = 0.0094). We conclude that with the present-day technology, ASE differences between individuals and between cases and controls are too subtle to be used to assess CRC risk. More advanced technology is expected to resolve this issue as well as the low informativity caused by the limited heterozygosity of transcribed SNPs.

RelA/p65 functions to maintain cellular senescence by regulating genomic stability and DNA repair.

Nuclear factor (NF)-kappaB is a positive regulator of tumour development and progression, but how it functions in normal cells leading to oncogenesis is not clear. As cellular senescence has proven to be an intrinsic tumour suppressor mechanism that cells must overcome to establish deregulated growth, we used primary fibroblasts to follow NF-kappaB function in cells transitioning from senescence to subsequent immortalization. Our findings show that RelA/p65(-/-) murine fibroblasts immortalize at considerably faster rates than RelA/p65(+/+) cells. The ability of RelA/p65(-/-) fibroblasts to escape senescence earlier is due to their genomic instability, characterized by high frequencies of DNA mutations, gene deletions and gross chromosomal translocations. This increase in genomic instability is closely related to a compromised DNA repair that occurs in both murine RelA/p65(-/-) fibroblasts and tissues. Significantly, these results can also be duplicated in human fibroblasts lacking NF-kappaB. Altogether, our findings present a fresh perspective on the role of NF-kappaB as a tumour suppressor, which acts in pre-neoplastic cells to maintain cellular senescence by promoting DNA repair and genomic stability.

Infrequent detection of germline allele-specific expression of TGFBR1 in lymphoblasts and tissues of colon cancer patients.

Recently, germline allele-specific expression (ASE) of the gene encoding for transforming growth factor-beta type I receptor (TGFBR1) has been proposed to be a major risk factor for cancer predisposition in the colon. Germline ASE results in a lowered expression of one of the TGFBR1 alleles (>1.5-fold), and was shown to occur in approximately 20% of informative familial and sporadic colorectal cancer (CRC) cases. In the present study, using the highly quantitative pyrosequencing technique, we estimated the frequency of ASE in TGFBR1 in a cohort of affected individuals from familial clusters of advanced colon neoplasias (cancers and adenomas with high-grade dysplasia), and also from a cohort of individuals with sporadic CRCs. Cases were considered positive for the presence of ASE if demonstrating an allelic expression ratio <0.67 or >1.5. Using RNA derived from lymphoblastoid cell lines, we find that of 46 informative Caucasian advanced colon neoplasia cases with a family history, only 2 individuals display a modest ASE, with allelic ratios of 1.65 and 1.73, respectively. Given that ASE of TGFBR1, if present, would likely be more pronounced in the colon compared with other tissues, we additionally determined the allele ratios of TGFBR1 in the RNA derived from normal-appearing colonic mucosa of sporadic CRC cases. We, however, found no evidence of ASE in any of 44 informative sporadic cases analyzed. Taken together, we find that germline ASE of TGFBR1, as assayed in lymphoblastoid and colon epithelial cells of colon cancer patients, is a relatively rare event.

Germline allele-specific expression of TGFBR1 confers an increased risk of colorectal cancer.

Much of the genetic predisposition to colorectal cancer (CRC) in humans is unexplained. Studying a Caucasian-dominated population in the United States, we showed that germline allele-specific expression (ASE) of the gene encoding transforming growth factor-beta (TGF-beta) type I receptor, TGFBR1, is a quantitative trait that occurs in 10 to 20% of CRC patients and 1 to 3% of controls. ASE results in reduced expression of the gene, is dominantly inherited, segregates in families, and occurs in sporadic CRC cases. Although subtle, the reduction in constitutive TGFBR1 expression alters SMAD-mediated TGF-beta signaling. Two major TGFBR1 haplotypes are predominant among ASE cases, which suggests ancestral mutations, but causative germline changes have not been identified. Conservative estimates suggest that ASE confers a substantially increased risk of CRC (odds ratio, 8.7; 95% confidence interval, 2.6 to 29.1), but these estimates require confirmation and will probably show ethnic differences.