16p13.11 microdeletion uncovers loss-of-function of a MYH11 missense variant in a patient with megacystis-microcolon-intestinal-hypoperistalsis syndrome.

Megacystis-microcolon-intestinal hypoperistalsis syndrome (MMIHS), a rare condition that affects smooth muscle cells, is caused by biallelic null alleles in MYH11. We report on a girl with MMIHS in addition to growth hormone deficiency, central hypothyroidism and a tonically dilated pupil with accommodation deficit. Sanger sequencing and arrayCGH uncovered the novel heterozygous missense variant c.379C>T in MYH11 and a heterozygous 1.3 Mb deletion in 16q13.11 encompassing MYH11, respectively. Her mother carries the deletion, whereas her father is heterozygous for the c.379C>T p.(Pro127Ser) change. Proline 127 is crucial for the formation of the Adenosine triphosphate binding pocket of the MYH11 motor domain and molecular modeling indicated that p.Pro127Ser alters nucleotide binding properties. Thus, the unusual and complex clinical presentation of the patient results from compound heterozygosity for a 16p13.11 microdeletion including the entire MYH11 gene and a loss-of-function missense variant on the remaining MYH11 allele. In conclusion, we recommend genetic testing both for MYH11 sequence alterations and copy number imbalances in individuals with MMIHS and smooth muscle cell-associated abnormalities in additional organs, that is, multisystemic smooth muscle dysfunction.

Looking for the hidden mutation: Bannayan-Riley-Ruvalcaba syndrome caused by constitutional and mosaic 10q23 microdeletions involving PTEN and BMPR1A.

The PTEN hamartoma tumor syndrome (PHTS) is caused by heterozygous germline variants in PTEN. Here, we report two unrelated patients with juvenile polyposis, macrocephaly, intellectual disability, and hyperpigmented skin macules. Both patients were clinically suspected for the Bannayan-Riley-Ruvalcaba syndrome (BRRS), a PHTS subentity. By array-CGH analysis, we identified an interstitial 10q23.1q23.3 deletion in a buccal mucosa sample of Patient 1 that encompassed PTEN, BMPR1A, and KLLN, among others. In contrast, neither sequencing nor array-CGH analysis identified a pathogenic variant in PTEN or BMPR1A in a blood sample of Patient 2. However, in a surgical specimen of the thyroid gland high-level mosaicism for a 10q23.2q23.3 deletion was observed. Additionally, the pathogenic PTEN variant c.956_959delCTTT p.(Thr319LysfsTer24) was detected in his thyroid tissue. The frame shift variant was neither detected in the patient's blood nor in his buccal mucosa sample. Low-level mosaicism for the microdeletion was identified in a buccal swap sample, and reanalysis of the blood sample suggested marginal-level mosaicism for deletion. The 10q23.2q23.3 deletion mosaicism was also identified in a subsequently resected colonic polyp. Thus, in both cases, the diagnosis of a 10q23 deletion syndrome, which clinically presented as BRRS, was established. Overall, the study expands the BRRS spectrum and highlights the relevance of considering mosaicism in PHTS. We conclude that in all patients with a clear clinical suspicion of PHTS, in which genetic analyses of DNA from blood and buccal swap samples fail to identify causative genetic variants, genetic analyses of additional tissues are recommended.

A tandem duplication of BRCA1 exons 1-19 through DHX8 exon 2 in four families with hereditary breast and ovarian cancer syndrome.

PURPOSE: The purpose of this study is to characterize a novel structural variant, a large duplication involving exons 1-19 of the BRCA1 gene in four independent families, and to provide diagnostically valuable information including the position of the breakpoints as well as clues to its clinical significance. METHODS: The duplication of exons 1-19 of the BRCA1 gene was initially detected by routine laboratory testing including MLPA analysis and next generation sequencing. For detailed characterization we performed array-comparative genome hybridization analysis, fluorescent in situ hybridization, next generation mapping, and long-distance PCR for break-point sequencing. RESULTS: Our data revealed a tandem duplication on chromosome 17 that encompassed 357 kb and included exons 1-19 of the BRCA1 gene and the genes NBR2, NBR1, TMEM106A, LOC100130581, ARL4D, MIR2117 up to parts of the DHX8 gene. This structural variant appeared as a tandem duplication with breakpoints in intron 19 of the BRCA1 gene and in intron 3 of the DHX8 gene (HGVS:chr17(hg19):g.41210776_41568516dup). Segregation analysis indicated that this structural rearrangement is phased in trans with a known pathogenic exon deletion of the BRCA1 gene in one family. CONCLUSIONS: The copy number variation initially recognized as duplication of exon 1-19 of the BRCA1 gene by MLPA analysis is a structural variation with breakpoints in the BRCA1 and DHX8 genes. Although currently to be classified as a variant of unknown significance, our family data indicates that this duplication may be a benign variation or at least of markedly reduced penetrance since it occurs in trans with another known fully pathogenic variant in the BRCA1 gene.

Gilbert syndrome redefined: a complex genetic haplotype influences the regulation of glucuronidation.

UNLABELLED: Gilbert syndrome (GS) is characterized by intermittent unconjugated hyperbilirubinemia without structural liver damage, affecting about 10% of the white population. In GS the UGT1A1*28 variant reduces bilirubin conjugation by 70% and is associated with irinotecan and protease inhibitor side effects. The aim of this study was to characterize potential in vivo consequences of UGT1A gene variability in GS. Three hundred GS patients (UGT1A1*28 homozygous) and 249 healthy blood donors (HBD) were genotyped for UGT1A (UGT1A1*28, UGT1A3-66 T>C, UGT1A6*3a, UGT1A7*3) and transporter single nucleotide polymorphisms (SNPs) (SCLO1B1 p.V174A, SCLO1B1 p.N130D, ABCC2 p.I1324I, ABCC2-24 UTR) using TaqMan-5'-nuclease-assays. A humanized transgenic UGT1A-SNP and corresponding wildtype mouse model were established carrying the GS-associated UGT1A variant haplotype. UGT1A transcript and protein expression, and transcriptional activation were studied in vivo. Homozygous UGT1A1*28 GS individuals were simultaneously homozygous for UGT1A3-66 T>C (91%), UGT1A6*2a (77%), and UGT1A7*3 (77%). Seventy-six percent of GS and only 9% of HBD were homozygous for the variant haplotype spanning four UGT1A genes. SCLO1B1 and ABCC2 SNPs showed no differences. In transgenic humanized UGT1A SNP and wildtype mice this UGT1A haplotype led to lower UGT1A messenger RNA (mRNA) expression and UGT1A protein synthesis. UGT1A transcriptional activation by dioxin, phenobarbital, and endotoxin was significantly reduced in SNP mice. CONCLUSION: Our data redefine the genetic basis behind GS. In vivo data studying the genotype present in 76% of GS individuals suggest that transcription and transcriptional activation of glucuronidation genes responsible for conjugation and detoxification is directly affected, leading to lower responsiveness. This study suggests that GS should be considered a potential risk factor for drug toxicity.

Prevalence of HSPB6 gene variants in peripartum cardiomyopathy: Data from the German PPCM registry.

BACKGROUND: Heat shock protein family B (small) member 6 (HSPB6) mediates cardioprotective effects against stress-induced injury. In humans two gene variants of HSPB6 have been identified with a prevalence of 1% in patients with dilated cardiomyopathy (DCM). Peripartum cardiomyopathy (PPCM) is a potentially life-threatening heart disease of unknown etiology in previously healthy women of whom 16-20% of PPCM carry gene variants associated with cardiomyopathy. This study was designed to analyze the prevalence of pathogenic HSPB6 gene variants in PPCM. METHODS AND RESULTS: Whole-exome sequencing was performed in whole blood samples of PPCM patients (n = 65 PPCM patients from the German PPCM registry) and screened subsequently for HSPB6 gene variants. In this PPCM cohort one PPCM patient carries a HSPB6 gene variant of uncertain significance (VUS), which was not associated with changes in the amino acid sequence and no likely pathogenic or pathogenic variants were detected. CONCLUSION: HSPB6 gene variants did not occur more frequently in a cohort of PPCM patients from the German PPCM registry, compared to DCM patients. Genetic analyses in larger cohorts and in cohorts of different ethiologies of PPCM patients are needed to address the role of the genetic background in the pathogenesis of PPCM.

Constitutional trisomy 8p11.21-q11.21 mosaicism: a germline alteration predisposing to myeloid leukaemia.

Juvenile myelomonocytic leukaemia (JMML) is a unique myeloproliferative disorder of early childhood. Frequently, mutations in NRAS, KRAS, PTPN11, NF1 or CBL are found in these patients. Monosomy 7 is the most common cytogenetic aberration. To identify submicroscopic genomic copy number alterations, 20 JMML samples were analysed by comparative genomic hybridization. Ten out of 20 samples displayed additional submicroscopic alterations. In two patients, an almost identical gain of chromosome 8 was identified. In both patients, fluorescence in situ hybridization confirmed a constitutional partial trisomy 8 mosaic (cT8M). A survey on 27 cT8M patients with neoplasms showed that 21 had myeloid malignancies, and five of these had a JMML. Notably, the region gained in our cases is the smallest gain of chromosome 8 reported in cT8M cases with malignancies so far. Our results dramatically reduce the critical region to 8p11.21q11.21 harbouring 31 protein coding genes and two non-coding RNAs, e.g. MYST3, IKBKB, UBE2V2, GOLGA7, FNTA and MIR486--a finding with potential implications for the role of somatic trisomy 8 in myeloid malignancies. Further investigations are required to more comprehensively determine how constitutional partial trisomy 8 mosaicisms may contribute to leukaemogenesis in different mutational subtypes of JMML and other myeloid malignancies.

PMS2 inactivation by a complex rearrangement involving an HERV retroelement and the inverted 100-kb duplicon on 7p22.1.

Biallelic PMS2 mutations are responsible for more than half of all cases of constitutional mismatch repair deficiency (CMMRD), a recessively inherited childhood cancer predisposition syndrome. The mismatch repair gene PMS2 is partly embedded within one copy of an inverted 100-kb low-copy repeat (LCR) on 7p22.1. In an individual with CMMRD syndrome, PMS2 was found to be homozygously inactivated by a complex chromosomal rearrangement, which separates the 5'-part from the 3'-part of the gene. The rearrangement involves sequences of the inverted 100-kb LCR and a human endogenous retrovirus element and may be associated with an inversion that is indistinguishable from the known inversion polymorphism affecting the ~0.7-Mb sequence intervening the LCR. Its formation is best explained by a replication-based mechanism (RBM) such as fork stalling and template switching/microhomology-mediated break-induced replication (FoSTeS/MMBIR). This finding supports the hypothesis that the inverted LCR can not only facilitate the formation of the non-allelic homologous recombination-mediated inversion polymorphism but it also promotes the occurrence of more complex rearrangements that can be associated with a large inversion, as well, but are mediated by a RBM. This further suggests that among the inversion polymorphism on 7p22.1, more complex rearrangements might be hidden. Furthermore, as the locus is embedded in a common fragile site (CFS) region, this rearrangement also supports the recently raised hypothesis that CFS sequence motifs may facilitate replication-based rearrangement mechanisms.

DNA-based genetic testing is rising steeply in a national health care system with open access to services: a survey of genetic test use in Germany, 1996-2002.

The extent to which the fast-growing body of genetic knowledge is transferred into everyday clinical practice has nowhere been assessed in a systematic way. Available quantitative analyses of DNA-based genetic test provision and uptake rates are all concerned with specific test programs. The German health-care system is ideally suited for a more general approach, because it is highly flexible regarding access to services, thus permitting quick adjustments to sudden changes in particular subfields of medicine such as genetic testing. We have measured the amount of genetic service provision in Germany between 1996 and 2002 by making use of the central database of the German national health-care system and by inquiring with private health insurance. We can document a three-fold increase of DNA-based testing in the time period 1996-2002, whereas cytogenetic analyses and genetic counseling have remained constant. The growing body of genetic knowledge does indeed seem to be transferred into medical practice at an increasing rate, and the uptake rates are largely in proportion to test offers. DNA-based testing appears to be focussed on disease-associated germ-line alterations.

Sequential anti-cytomegalovirus response monitoring may allow prediction of cytomegalovirus reactivation after allogeneic stem cell transplantation.

BACKGROUND: Reconstitution of cytomegalovirus-specific CD3(+)CD8(+) T cells (CMV-CTLs) after allogeneic hematopoietic stem cell transplantation (HSCT) is necessary to bring cytomegalovirus (CMV) reactivation under control. However, the parameters determining protective CMV-CTL reconstitution remain unclear to date. DESIGN AND METHODS: In a prospective tri-center study, CMV-CTL reconstitution was analyzed in the peripheral blood from 278 patients during the year following HSCT using 7 commercially available tetrameric HLA-CMV epitope complexes. All patients included could be monitored with at least CMV-specific tetramer. RESULTS: CMV-CTL reconstitution was detected in 198 patients (71%) after allogeneic HSCT. Most importantly, reconstitution with 1 CMV-CTL per µl blood between day +50 and day +75 post-HSCT discriminated between patients with and without CMV reactivation in the R+/D+ patient group, independent of the CMV-epitope recognized. In addition, CMV-CTLs expanded more daramtaically in patients experiencing only one CMV-reactivation than those without or those with multiple CMV reactivations. Monitoring using at least 2 tetramers was possible in 63% (n = 176) of the patients. The combinations of particular HLA molecules influenced the numbers of CMV-CTLs detected. The highest CMV-CTL count obtained for an individual tetramer also changed over time in 11% of these patients (n = 19) resulting in higher levels of HLA-B*0801 (IE-1) recognizing CMV-CTLs in 14 patients. CONCLUSIONS: Our results indicate that 1 CMV-CTL per µl blood between day +50 to +75 marks the beginning of an immune response against CMV in the R+/D+ group. Detection of CMV-CTL expansion thereafter indicates successful resolution of the CMV reactivation. Thus, sequential monitoring of CMV-CTL reconstitution can be used to predict patients at risk for recurrent CMV reactivation.

Novel CHD7 mutations contributing to the mutation spectrum in patients with CHARGE syndrome.

CHARGE syndrome is an autosomal dominant inherited multiple malformation disorder typically characterized by coloboma, choanal atresia, hypoplastic semicircular canal, cranial nerve defects, cardiovascular malformations and ear abnormalities. Mutations in the chromodomain helicase DNA-binding protein 7 (CHD7) gene are the major cause of CHARGE syndrome. Mutation analysis was performed in 18 patients with firm or tentative clinical diagnosis of CHARGE syndrome. In this study eight mutations distributed across the gene were found. Five novel mutations - one missense (c.2936T > C), one nonsense (c.8093C > A) and three frameshift mutations (c.804_805insAT, c.1757_1770del14, c.1793delA) - were identified. As far as familial data were available these mutations were found to have arisen de novo. Comparison of the clinical features of patients with the same mutation demonstrates that expression of the phenotype is highly variable. The mutation detection rate in this study was 44.4% in patients with a clinically established or suspected diagnosis of CHARGE syndrome.

Correlation of donor leukocyte chimerism with pulmonary allograft survival after immunosuppressive drug withdrawal in a porcine model.

BACKGROUND: This study was designed to analyze the role of postoperative donor cell chimerism for the induction and maintenance of transplantation tolerance in a porcine lung transplantation model. METHODS: Left-sided single lung transplantation from major histocompatibility mismatched male donors was performed in 27 female minipigs. All received a 28-day course of pharmacologic immunosuppression using various agents, some in combination with preoperative irradiation. Groups for eventual analysis were strictly defined by outcome, that is, pigs with acute rejection before postoperative day 178 (n=16) were allocated into one group, long-term surviving animals (n=11) into the other. Peripheral blood chimerism was monitored by flow cytometry and real-time polymerase chain reaction. Intragraft chimerism was detected from bronchoalveolar lavage fluid (BALF) by fluorescent in situ hybridization. RESULTS: Blood chimerism peaked 1 hour after transplantation and was significantly higher in the group of long-term survivors at that time. Thereafter chimerism rapidly decreased, but tended to remain higher in long-term survivors. In case of acute rejection donor cells were lost, but remained detectable for up to 36 postoperative months in tolerant animals. In BALF, the percentage of male nuclei was equally high under immunosuppression in both groups. Rejecting animals showed a rapid decrease of Y-bearing cells in BALF after drug withdrawal and an almost complete loss when acute rejection occurred. In tolerant pigs, intragraft chimerism remained detectable throughout the follow-up. CONCLUSIONS: This study demonstrates a clear correlation of donor leukocyte chimerism with long-term allograft survival in a porcine allogeneic lung transplantation model.

Multi-exon out of frame deletion of the FBN1 gene leading to a severe juvenile onset cardiovascular phenotype in Marfan syndrome.

Marfan syndrome is caused by mutations in fibrillin-1, a large gene spanning approximately 200 kb of genomic DNA on chromosome 15q21. So far, more than 600 different mutations have been identified, accounting for 60-90% of all Marfan syndrome cases, the vast majority being single nucleotide exchanges as well as small deletions and insertions. Only four major rearrangements have been described in the literature so far. We have screened 11 individuals fulfilling the diagnostic criteria of Marfan syndrome but negative for point mutations in the fibrillin-1 gene by SSCP and/or direct sequencing, for large rearrangements. We report here the largest known de novo and out of frame deletion in the fibrillin-1 gene in a patient fulfilling the diagnostic criteria of Marfan syndrome. We identified the deletion breakpoints at the genomic and transcript levels and studied the expression of the mutated allele at the transcript and protein level. We conclude that large rearrangements may account for a non-negligible proportion of all Marfan cases.

Reciprocal translocation between Y chromosome long arm euchromatin and the short arm of chromosome 1.

A case with an apparently balanced reciprocal translocation between the long arm of the Y chromosome and the short arm of chromosome 1 t(Y;1)(q11.2;p34.3) is described. The translocation was found in a phenotypically normal male ascertained by infertility and presenting for intra-cytoplasmatic sperm injection treatment. Histological examination of testicular biopsies revealed spermatogenic failure. Chromosome painting with probes for chromosome 1 and for the euchromatic part of the Y chromsome confirmed the translocation of euchromatic Y chromosomal material onto the short arm of chromosome 1 and of a substantial part of the short arm of chromosome 1 onto the Y chromosome. Among the Y/autosome translocations, the rearrangements involving long arm euchromatin of the Y chromosome are relatively rare and mostly associated with infertility. Microdeletion screening at the azoospermia locus revealed no deletions, suggesting another mechanism causing infertility in this translocation carrier.