Diagnosis of monogenic liver diseases in childhood by next-generation sequencing.

Next-generation sequencing (NGS) has opened up novel diagnostic opportunities for children with unidentified, but suspected inherited diseases. We describe our single-center experience with NGS diagnostics in standard clinical scenarios in pediatric hepatology. We investigated 135 children with suspected inherited hepatopathies, where initially no causative pathogenic variant had been identified, with an amplicon-based NGS panel of 21 genes associated with acute and chronic hepatopathies. In 23 of these patients, we detected pathogenic or likely pathogenic variants in 10 different genes. We present 6 novel variants. A total of 14 of these patients presented with the characteristic phenotype of the related hepatopathy. Nine patients showed only few or atypical clinical symptoms or presented with additional signs. In another 13 out of 135 cases, we detected variants of unknown significance (VUS) in 9 different genes. Only 2 of these patients showed characteristic phenotypes conclusive with the detected variants, whereas 11 patients showed unspecific or atypical phenotypes. Our multi-gene panel is a fast and comprehensive tool to diagnose inherited pediatric hepatopathies. We also illustrate the challenge of dealing with genetic variants and highlight arising clinical questions, especially in patients with atypical phenotypes.

Human BRCA1-associated breast cancer: no increase in numerical chromosomal instability compared to sporadic tumors.

BRCA1 is a major gatekeeper of genomic stability. Acting in multiple central processes like double-strand break repair, centrosome replication, and checkpoint control, BRCA1 participates in maintaining genomic integrity and protects the cell against genomic instability. Chromosomal instability (CIN) as part of genomic instability is an inherent characteristic of most solid tumors and is also involved in breast cancer development. In this study, we determined the extent of CIN in 32 breast cancer tumors of women with a BRCA1 germline mutation compared to 62 unselected breast cancers. We applied fluorescence in situ hybridization (FISH) with centromere-specific probes for the chromosomes 1, 7, 8, 10, 17, and X and locus-specific probes for 3q27 (BCL6), 5p15.2 (D5S23), 5q31 (EGR1), 10q23.3 (PTEN), and 14q32 (IGH@) on formalin-fixed paraffin-embedded tissue microarray sections. Our hypothesis of an increased level of CIN in BRCA1-associated breast cancer could not be confirmed by this approach. Surprisingly, we detected no significant difference in the extent of CIN in BRCA1-mutated versus sporadic tumors. The only exception was the CIN value for chromosome 1. Here, the extent of CIN was slightly higher in the group of sporadic tumors.

Generation and characterization of a transgenic mouse with a functional human TSPY.

To generate an animal model that is suitable for the analysis of regulation and expression of human testis-specific protein, Y-encoded TSPY, a transgenic mouse line, TgTSPY9, harboring a complete structural human TSPY gene was generated. Fluorescence in situ hybridization and Southern analyses show that approximately 50 copies of the human TSPY transgene are integrated at a single chromosomal site that maps to the distal long arm of the Y chromosome. The transgene is correctly transcribed and spliced according to the human pattern and is mainly expressed in testicular tissue, with spermatogonia and early primary spermatocytes (leptotene and zygotene) as expressing germ cells. TSPY transgenic mice are phenotypically normal, and spermatogenesis is neither impaired nor enhanced by the human transgene. The present study shows that a human TSPY gene integrated into the mouse genome follows the human expression pattern although murine tspy had lost its function in rodent evolution millions of years ago.

Spectrum of ATM gene mutations in a hospital-based series of unselected breast cancer patients.

Blood relatives of patients with the inherited disease ataxia telangiectasia (A-T) have an increased susceptibility for breast cancer. We therefore looked for sequence alterations of the ATM gene in a large hospital-based series of unselected breast cancer patients. The whole ATM coding sequence was analyzed in genomic DNA samples from a core group of 192 consecutive breast cancer cases to define the spectrum of ATM gene mutations. Common sequence alterations were then screened in the whole series of 1000 breast cancer patients and in 500 random individuals. In the core group, 21 distinct sequence alterations were identified throughout the ATM coding region, and 1 common splicing mutation was uncovered in intron 10. Almost half of the breast cancer patients (46%) were heterozygotes for 1 of 16 different amino acid substitutions, and three patients (1.6%) carried a truncating mutation. These data indicate that approximately 1 in 50 German breast cancer patients is heterozygous for an A-T-causing mutation. In our extended series, the most common A-T mutation 1066-6T-->G was disclosed in 7 of 1000 (0.7%) breast cancer patients. Transcript analyses indicated that the loss of exon 11 in the ATM mRNA was the pathogenic consequence of this splicing mutation, which produced a <10% of full-length ATM mRNA and ATM protein in a homozygous A-T patient. We also found an excess of rare missense substitutions in the breast cancer cohort compared with random individuals (7.9% versus 5.3% of alleles; odds ratio = 1.6; P < 0.01). One missense substitution, S707P in exon 15, was two times more frequent in breast cancer patients (odds ratio = 2.4; 95% confidence interval, 1.0-5.8) and five times more frequent in patients with bilateral disease than in random individuals (P < 0.001). We conclude that a large variety of distinct ATM mutations and variants exist among breast cancer patients, some of which can contribute to the etiology and progression of the malignancy. Screening for frequent A-T mutations such as the 1066-6-->G splice site substitution can be effective to prospectively identify A-T heterozygotes in an unselected cancer patient population.

Mutations of the BRCA1 and BRCA2 genes in patients with bilateral breast cancer.

Mutations of the BRCA1 or BRCA2 genes have been shown to strongly predispose towards the development of contralateral breast cancer in patients from large multi-case families. In order to test the hypothesis that BRCA1 and BRCA2 mutations are more frequent in patients with bilateral breast cancer, we have investigated a hospital-based series of 75 consecutive patients with bilateral breast cancer and a comparison group of 75 patients with unilateral breast cancer, pairwise matched by age and family history, for mutations in the BRCA1 and BRCA2 genes. Five frameshift deletions (517delGT in BRCA1; 4772delA, 5946delCT, 6174delT and 8138del5 in BRCA2) were identified in patients with bilateral disease. No further mutations, apart from polymorphisms and 3 rare unclassified variants, were found after scanning the whole BRCA1 and BRCA2 coding sequence. Three pathogenic BRCA1 mutations (Cys61Gly, 3814del5, 5382insC) were identified in the group of patients with unilateral breast cancer. The frequencies of common BRCA1 and BRCA2 missense variants were not different between the 2 groups. In summary, we did not find a significantly increased prevalence of BRCA1 and BRCA2 mutations in a hospital-based cohort of German patients with bilateral breast cancer. We conclude that bilaterality of breast cancer on its own is not strongly associated with BRCA1 and BRCA2 mutations when adjusted for age and family history. The high frequency of bilateral disease in multi-case breast cancer families may be due to a familial aggregation of additional susceptibility factors modifying the penetrance of BRCA1 and BRCA2 mutations.

Silencing of the Y-chromosomal gene tspy during murine evolution.

We have studied the process of tspy gene silencing in murine evolution. We have isolated functional tspy sequences from Apodemus agrarius, A. sylvaticus, A. flavicollis, and Mus platythrix (subgenus Pyromys) and nonfunctional tspy sequences from species of the subgenus Mus. We present two alternative models as to how tspy may have lost its function in the murine lineage.

TSPY variants in six loci on the human Y chromosome.

We have studied the structure, organization, and evolution of the human TSPY gene family by mapping three sequence variants identified through RT-PCR analysis onto genomic clones derived from two different YAC contigs. TSPY gene family members occur in at least six locations on the human Y chromosome, and each cluster contains a unique combination of variants. Our data further suggest that an 18-bp tandem duplication found in TSPY exon 1 originated from an unequal sister chromatid exchange between two tandemly arranged TSPY clusters.

Molecular evolution of the murine tspy genes.

Molecular aspects of murine evolution were studied by sequencing, and subsequently comparing, introns of the Y-chromosomal tspy genes from Apodemus agrarius, A. sylvaticus, A. flavicollis, Mus platythrix (subgenus Pyromys), M. booduga (subgenus Leggada), and from species of the subgenus Mus, including M. cervicolor, M. macedonicus and M. spretus. Estimates of nucleotide substitution rates in these lineages were in perfect agreement with phylogenetic data previously published by She et al. (1990), Catzeflis et al. (1992; 1993), and Lyon et al. (1996). The only exception was provided by a comparatively late divergence of M. spretus and M. macedonicus. Our data also suggest that M. booduga diverged from the subgenus Mus about 3 Myr ago.

Frequency of BRCA1 mutation 5382insC in German breast cancer patients.

OBJECTIVE: The aim of the study was to determine the frequency of the BRCA1 mutation 5382insC in German breast cancer patients with and without prior knowledge of a family history of breast cancer. METHODS: Two groups of breast cancer patients were tested for the presence or absence of the 5382insC mutation using a PCR primer mismatch assay. A sample of 248 patients unrelated by genealogy was selected based on a history of breast and/or ovarian cancer in the families. In addition, a population-based sample of 800 unselected breast cancer patients was included in the analysis. Three intragenic DNA markers D17S1323, D17S1322, and D17S855, located at BRCA1 introns 12, 19, and 20, respectively, were utilized for allelic association studies as well as for haplotype analysis in 4 breast/ovarian cancer families. RESULTS: The 5382insC mutation was identified in 10/248 (4.0%) familial breast cancer patients and in 8/800 (1.0%) unselected cases. Allelic association studies and haplotype analysis revealed an association of allele Nos. "6" at D17S1323 (chi2 value = 9.34, P = 0.007), "5" at D17S1322 (chi2 value = 3.62, P = 0.171), and "4" at D17S855 (chi2 value = 11.34, P = 0. 002) with the mutation 5382insC. CONCLUSION: 5382insC constitutes a frequent BRCA1 mutation in German breast cancer patients. The significant allelic association between this mutation and two intragenic DNA markers (D17S1323, D17S855) and the elevated allele frequency at marker D17S1322 suggest an ancient founder in the German breast cancer population. The PCR primer mismatch assay described herein provides a rapid and reliable detection method for the recurrent 5382insC mutation and will be useful for the analysis of large breast cancer populations.

Characterization of ATM gene mutations in 66 ataxia telangiectasia families.

Ataxia telangiectasia (AT) is an autosomal recessive disease characterized by neurological and immunological symptoms, radiosensitivity and cancer predisposition. The gene mutated in AT, designated the ATM gene, encodes a large protein kinase with a PI-3 kinase-related domain. In this study, we investigated the mutational spectrum of the ATM gene in a cohort of AT patients living in Germany. We amplified and sequenced all 66 exons and the flanking untranslated regions from genomic DNA of 66 unrelated AT patients. We identified 46 different ATM mutations and 26 sequence polymorphisms and variants scattered throughout the gene. A total of 34 mutations have not been described in other populations. Seven mutations occurred in more than one family, but none of these accounted for more than five alleles in our patient group. The majority of the mutations were truncating, confirming that the absence of full-length ATM protein is the most common molecular basis of AT. Transcript analyses demonstrated single exon skipping as the consequence of most splice site substitutions, but a more complex pattern was observed for two mutations. Immunoblot studies of cell lines carrying ATM missense substitutions or in-frame deletions detected residual ATM protein in four cases. One of these mutations, a valine deletion proximal to the kinase domain, resulted in ATM protein levels >20% of normal in an AT lymphoblastoid cell line. In summary, our results survey and characterize a plethora of variations in the ATM gene identified by exon scanning sequencing and indicate a high diversity of mutations giving rise to AT in a non-isolated population.

Detection of 100% of the CFTR mutations in 63 CF families from Tyrol.

We identified 100% of the CFTR gene mutations, including three novel mutations, in 126 unrelated cystic fibrosis chromosomes from Tyrol, Austria. The frequency of the major mutation deltaF508 (74.6%) was not significantly different in Tyrolian CF-patients than in patients from Bavaria (71.0%) and Middle- and Northern Germany (71.9%), but was significantly higher than in patients from Styria (58.1%) or Northern Italy (47.6%). Interestingly, the distribution of the next most frequent mutations, R1162X (8.7%) 2183AA-->G, 2789+5G-->A and G542X (2.4% each), was more similar to the distribution of these mutations among CF-patients from Northern Italy than to those from Styria, Bavaria or Middle- and Northern Germany. Nine further mutations occurred once or twice. One of these, the missense mutation M1101K, is rare worldwide but very frequent in the Hutterite brethren, a small founder population which came from Southern Austria to Northern America. Three other different mutations (deltaL453, 1874insT and 4108delT) were present in single Tyrolian families and have not been described before. The identification of 100% of CFTR gene mutations in a particular CF population demonstrates the power of genetic analysis for the diagnosis and counselling of CF families in this restricted geographical area of Austria. Our study provides evidence for a closer genetic relation between CF patients from Tyrol and those from Bavaria or Middle- and Northern Germany as well as Northern Italy, than between CF patients from the two Austrian states Tyrol and Styria.