To identify novel risk variants for chronic lymphocytic leukemia (CLL) we conducted a genome-wide association study of 299,983 tagging SNPs, with validation in four additional series totaling 2,503 cases and 5,789 controls. We identified four risk loci for CLL at 2q37.3 (rs757978, FARP2; odds ratio [OR] = 1.39; P = 2.11 x 10-9), 8q24.21 (rs2456449; OR = 1.26; P = 7.84 x 10-10), 15q21.3 (rs7169431; OR = 1.36; P = 4.74 x 10-7) and 16q24.1 (rs305061; OR = 1.22; P = 3.60 x 10-7). There was also evidence for risk loci at 15q25.2 (rs783540, CPEB1; OR = 1.18; P = 3.67 x 10-6) and 18q21.1 (rs1036935; OR = 1.22; P = 2.28 x 10-6). These data provide further evidence for genetic susceptibility to this B-cell hematological malignancy.

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Premature ovarian failure (POF) is characterized by hypergonadotropic hypogonadism and amenorrhea before the age of 40. The condition has a heterogeneous background but genetic factors are demonstrated by the occurrence of familial cases. We identified a mother and daughter with POF both of whom carry an X;autosome translocation [t(X;11)(q24;q13)]. RNA expression studies of genes flanking the X-chromosome breakpoint revealed that both patients have reduced expression levels of the gene Progesterone Receptor Membrane Component-1 (PGRMC1). Mutation screening of 67 females with idiopathic POF identified a third patient with a missense mutation (H165R) located in the cytochrome b5 domain of PGRMC1. PGRMC1 mediates the anti-apoptotic action of progesterone in ovarian cells and it acts as a positive regulator of several cytochrome P450 (CYP)-catalyzed reactions. The CYPs are critical for intracellular sterol metabolism, including biosynthesis of steroid hormones. We show that the H165R mutation associated with POF abolishes the binding of cytochrome P450 7A1 (CYP7A1) to PGRMC1. In addition, the missense mutation attenuates PGRMC1's ability to mediate the anti-apoptotic action of progesterone in ovarian cells. These findings suggest that mutant or reduced levels of PGMRC1 may cause POF through impaired activation of the microsomal cytochrome P450 and increased apoptosis of ovarian cells.

X-linked mental retardation (XLMR) affects one in 600 males and is highly heterogeneous. We describe here a 29-year-old woman with severe nonsyndromic mental retardation and a balanced reciprocal translocation between chromosomes X and 15 [46,XX,t(X;15)(q13.3;cen)]. Methylation studies showed a 100% skewed X-inactivation in patient-derived lymphocytes indicating that the normal chromosome X is retained inactive. Physical mapping of the breakpoints localised the Xq13.3 breakpoint to within 3.9 kb of the first exon of the ZDHHC15 gene encoding a zinc-finger and a DHHC domain containing product. Expression analysis revealed that different transcript variants of the gene are expressed in brain. ZDHHC15-specific RT-PCR analysis on lymphocytes from the patient revealed an absence of ZDHHC15 transcript variants, detected in control samples. We suggest that the absence of the ZDHHC15 transcripts in this patient contributes to her phenotype, and that the gene is a strong candidate for nonsyndromic XLMR.

Screening for gene copy-number alterations (CNAs) has improved by applying genome-wide microarrays, where SNP arrays also allow analysis of loss of heterozygozity (LOH). We here analyzed 10 chronic lymphocytic leukemia (CLL) samples using four different high-resolution platforms: BAC arrays (32K), oligonucleotide arrays (185K, Agilent), and two SNP arrays (250K, Affymetrix and 317K, Illumina). Cross-platform comparison revealed 29 concordantly detected CNAs, including known recurrent alterations, which confirmed that all platforms are powerful tools when screening for large aberrations. However, detection of 32 additional regions present in 2-3 platforms illustrated a discrepancy in detection of small CNAs, which often involved reported copy-number variations. LOH analysis using dChip revealed concordance of mainly large regions, but showed numerous, small nonoverlapping regions and LOH escaping detection. Evaluation of baseline variation and copy-number ratio response showed the best performance for the Agilent platform and confirmed the robustness of BAC arrays. Accordingly, these platforms demonstrated a higher degree of platform-specific CNAs. The SNP arrays displayed higher technical variation, although this was compensated by high density of elements. Affymetrix detected a higher degree of CNAs compared to Illumina, while the latter showed a lower noise level and higher detection rate in the LOH analysis. Large-scale studies of genomic aberrations are now feasible, but new tools for LOH analysis are requested.

The online version of this article has a Supplementary Appendix. BackgroundThe existence of multiple subsets of chronic lymphocytic leukemia expressing 'stereotyped' Bcell receptors implies the involvement of antigen(s) in leukemogenesis. Studies also indicate that 'stereotypy' may influence the clinical course of patients with chronic lymphocytic leukemia, for example, in subsets with stereotyped IGHV3-21 and IGHV4-34 B-cell receptors; however, little is known regarding the genomic profile of patients in these subsets. Design and MethodsWe applied 250K single nucleotide polymorphism-arrays to study copy-number aberrations and copy-number neutral loss-of-heterozygosity in patients with stereotyped IGHV3-21 (subset #2, n=29), stereotyped IGHV4-34 (subset #4, n=17; subset #16, n=8) and non-subset #2 IGHV3-21 (n=13) and non-subset #4/16 IGHV4-34 (n=34) patients. ResultsOver 90% of patients in subset #2 and non-subset #2 carried copy-number aberrations, whereas 75-76% of patients in subset #4 and subset #16 showed copy-number aberrations. Subset #2 and non-subset #2 patients also displayed a higher average number of aberrations compared to patients in subset #4. Deletion of 13q was the only known recurrent aberration detected in subset #4 (35%); this aberration was even more frequent in subset #2 (79%). del(11q) was more frequent in subset #2 and non-subset #2 (31% and 23%) patients than in subset #4 and nonsubset #4/16 patients. Recurrent copy-number neutral loss-of-heterozygosity was mainly detected on chromosome 13q, independently of B-cell receptor stereotypy. ConclusionsGenomic aberrations were more common in subset #2 and non-subset #2 than in subset #4. The particularly high frequency of del(11q) in subset #2 may be linked to the adverse outcome reported for patients in this subset. Conversely, the lower prevalence of copy-number aberrations and the absence of poor-prognostic aberrations in subset #4 may reflect an inherently low-proliferative disease, which would prevent accumulation of genomic alterations.Key words: chronic lymphocytic leukemia, stereotyped B-cell receptors, antigens, leukemogenesis. 'stereotyped' IGHV3-21 and IGHV4-34 B-cell receptors. Haematologica 2010;95(9):1519-1525. doi:10.3324/haematol.2009 This is an open-access paper. Citation: Marincevic M, Cahill N, Gunnarsson R, Isaksson A, Mansouri M, Göransson H, Rasmussen M, Jansson M, Ryan F, Karlsson K, Adami H-O, Davi F, Jurlander J, Juliusson G, Stamatopoulos K, and Rosenquist R. High-density screening reveals a different spectrum of genomic aberrations in chronic lymphocytic leukemia patients withHigh-density screening reveals a different spectrum of genomic aberrations in chronic lymphocytic leukemia patients with 'stereotyped' IGHV3-21 and IGHV4-34 B-cell receptors

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Screening for copy number alterations (CNA) has improved by applying genome wide microarrays, where SNP-arrays also allow analysis of loss of heterozygosity (LOH). Currently, comparisons of high resolution microarray platforms are few, thus we performed a study to evaluate the power of differently designed microarrays for copy number analysis and LOH. We here analyzed 10 diagnostic chronic lymphocytic leukemia (CLL) samples (five IGVH mutated and five IGVH unmutated) using four different high-resolution platforms: BAC-arrays (32K), oligonucleotide-arrays (185K, Agilent), and two SNP-arrays (250K, Affymetrix and 317K, Illumina). Comparison of copy number data showed that the platforms are concordant in terms of detecting large CNA, including the known recurrent alterations. Mono-allelic and bi-allelic loss of 13q14 (3 and 1 sample, respectively), mono-allelic loss of 11q (1 sample), trisomy 12 (2 samples) and mono-allelic loss of 17p (2 samples) were concordant in all platforms. These aberrations were validated with FISH, which in addition identified subclones with mono-allelic loss of 13q14 in two cases, only detected with the BAC platform, rendering a cut-off for the power of detecting subclones to approximately 25% of investigated cells. As expected, all poor prognostic aberrations were detected in patients carrying unmutated IGHV genes whereas four of five mutated samples were detected with mono-allelic loss of 13q14, as the only recurrent alteration. Furthermore, detection of small CNA were in many cases discordant between platforms. Therefore, we defined alterations identified by at least two platforms and identified 47 losses and 31 gains using this criterion. We are currently validating the presence of a number of these alterations using other techniques. Evaluation of LOH showed concordance for 86 regions between the Illumina and Affymetrix platforms. Of these regions 12 LOH coincided with CNA, leaving the remaining 74 as copy-neutral LOH. In conclusion, all platforms investigated are powerful tools for screening of CNA, however, since non-overlapping CNA were detected by individual platforms, we emphasize the importance of validating findings. Also, there is a cut-off for detecting subclones, here estimated to 25%. Genomic arrays will improve the detection of new recurrent aberrations, which may potentially refine the prognostic hierarchy established by FISH.