Aki Mustonen

A recurrent mutation in PALB2 in Finnish cancer families

BRCA1, BRCA2 and other known susceptibility genes account for less than half of the detectable hereditary predisposition to breast cancer. Other relevant genes therefore remain to be discovered. Recently a new BRCA2-binding protein, PALB2, was identified. The BRCA2–PALB2 interaction is crucial for certain key BRCA2 DNA damage response functions as well as its tumour suppression activity. Here we show, by screening for PALB2 mutations in Finland that a frameshift mutation, c.1592delT, is present at significantly elevated frequency in familial breast cancer cases compared with ancestry-matched population controls. The truncated PALB2 protein caused by this mutation retained little BRCA2-binding capacity and was deficient in homologous recombination and crosslink repair. Further screening of c.1592delT in unselected breast cancer individuals revealed a roughly fourfold enrichment of this mutation in patients compared with controls. Most of the mutation-positive unselected cases had a familial pattern of disease development. In addition, one multigenerational prostate cancer family that segregated the c.1592delT truncation allele was observed. These results indicate that PALB2 is a breast cancer susceptibility gene that, in a suitably mutant form, may also contribute to familial prostate cancer development.

Potential Novel Mechanism for Axenfeld-Rieger Syndrome: Deletion of a Distant Region Containing Regulatory Elements of PITX2

PURPOSE Mutations in PITX2 are associated with Axenfeld-Rieger syndrome (ARS), which involves ocular, dental, and umbilical abnormalities. Identification of cis-regulatory elements of PITX2 is important to better understand the mechanisms of disease. METHODS Conserved noncoding elements surrounding PITX2/pitx2 were identified and examined through transgenic analysis in zebrafish; expression pattern was studied by in situ hybridization. Patient samples were screened for deletion/duplication of the PITX2 upstream region using arrays and probes. RESULTS Zebrafish pitx2 demonstrates conserved expression during ocular and craniofacial development. Thirteen conserved noncoding sequences positioned within a gene desert as far as 1.1 Mb upstream of the human PITX2 gene were identified; 11 have enhancer activities consistent with pitx2 expression. Ten elements mediated expression in the developing brain, four regions were active during eye formation, and two sequences were associated with craniofacial expression. One region, CE4, located approximately 111 kb upstream of PITX2, directed a complex pattern including expression in the developing eye and craniofacial region, the classic sites affected in ARS. Screening of ARS patients identified an approximately 7600-kb deletion that began 106 to 108 kb upstream of the PITX2 gene, leaving PITX2 intact while removing regulatory elements CE4 to CE13. CONCLUSIONS These data suggest the presence of a complex distant regulatory matrix within the gene desert located upstream of PITX2 with an essential role in its activity and provides a possible mechanism for the previous reports of ARS in patients with balanced translocations involving the 4q25 region upstream of PITX2 and the current patient with an upstream deletion.

Clinical, enzymatic, and molecular genetic characterization of a biochemical variant type of argininosuccinic aciduria: prenatal and postnatal diagnosis in five unrelated families

A biochemical variant of argininosuccinate lyase deficiency, found in five individuals, is introduced. In comparison to classical patients, the variant cases of argininosuccinate lyase deficiency were characterized by residual enzyme activity as measured by the incorporation of [14C]citrulline into proteins. The five patients of different ethnic backgrounds presented with relatively mild clinical symptoms, variable age of onset, marked argininosuccinic aciduria and severe, but not complete, deficiency of argininosuccinate lyase. [14C]Citrulline incorporation into proteins, which is completely blocked in classical argininosuccinic aciduria, was only partially reduced in fibroblasts of these patients. Further investigation showed that previous standard conditions of the assay were not optimal. Higher concentrations of citrulline in the incubation medium strongly stimulated 14C incorporation in normal cells, but not in the patients; as a result, the relative incorporation level in the patients dropped to 6–28% compared to 18–75% of normal in the original procedure. Prenatal diagnosis was successfully performed in three of the families. Affected pregnancies were indicated by (partial) deficiency of [14C]citrulline incorporation in chorionic villi and/or increased levels of argininosuccinate in amniotic fluid. Analysis of the ASL gene in the five patients revealed a considerable allelic heterogeneity. Three novel mutations—R385C (2 patients), V178M and R379C—were detected in homozygous states, whereas one patient was compound heterozygous for the known mutations R193Q and Q286R. In conclusion, there are patients of different ethnic backgrounds who are characterized by residual activity of argininosuccinate lyase and who present with less severe clinical courses. In addition, we present an improved biochemical assay for accurate prenatal and postnatal diagnosis.

Prenatal diagnosis of Batten's disease

BACKGROUND Battens disease is the most common progressive encephalopathy of childhood in Western countries. The major mutation is a 1kb deletion, which is carried by 81% of Battens disease patients. We report on the use of direct gene analysis in the prenatal diagnosis of this disease. METHODS AND FINDINGS A Finnish woman with a son with Battens disease came for genetic counselling for her current pregnancy. Electron microscopy of a chorionic villus sample gave suggestive findings. We used PCR to look for the intragenic microsatellite marker D16S298; 96% of Finnish Battens disease patients carry allele 6 at this marker. The fetus and the affected son both carried the same high-risk genotype, 6/6. Both were homozygous for the 1 kb deletion. The pregnancy was terminated. Electron microscopy of the fetus showed typical Battens disease changes. INTERPRETATION We have successfully used direct gene analysis in the prenatal diagnosis of Battens disease.

Genome-wide search for breast cancer linkage in large Icelandic non-BRCA1/2 families

Introduction:A significant proportion of high-risk breast cancer families are not explained by mutations in known genes. Recent genome-wide searches (GWS) have not revealed any single major locus reminiscent of BRCA1 and BRCA2, indicating that still unidentified genes may explain relatively few families each or interact in a way obscure to linkage analyses. This has drawn attention to possible benefits of studying populations where genetic heterogeneity might be reduced. We thus performed a GWS for linkage on nine Icelandic multiple-case non-BRCA1/2 families of desirable size for mapping highly penetrant loci. To follow up suggestive loci, an additional 13 families from other Nordic countries were genotyped for selected markers.Methods:GWS was performed using 811 microsatellite markers providing about five centiMorgan (cM) resolution. Multipoint logarithm of odds (LOD) scores were calculated using parametric and nonparametric methods. For selected markers and cases, tumour tissue was compared to normal tissue to look for allelic loss indicative of a tumour suppressor gene.Results:The three highest signals were located at chromosomes 6q, 2p and 14q. One family contributed suggestive LOD scores (LOD 2.63 to 3.03, dominant model) at all these regions, without consistent evidence of a tumour suppressor gene. Haplotypes in nine affected family members mapped the loci to 2p23.2 to p21, 6q14.2 to q23.2 and 14q21.3 to q24.3. No evidence of a highly penetrant locus was found among the remaining families. The heterogeneity LOD (HLOD) at the 6q, 2p and 14q loci in all families was 3.27, 1.66 and 1.24, respectively. The subset of 13 Nordic families showed supportive HLODs at chromosome 6q (ranging from 0.34 to 1.37 by country subset). The 2p and 14q loci overlap with regions indicated by large families in previous GWS studies of breast cancer.Conclusions:Chromosomes 2p, 6q and 14q are candidate sites for genes contributing together to high breast cancer risk. A polygenic model is supported, suggesting the joint effect of genes in contributing to breast cancer risk to be rather common in non-BRCA1/2 families. For genetic counselling it would seem important to resolve the mode of genetic interaction.

Mutation analysis for prenatal diagnosis of hereditary tyrosinaemia type 1

Hereditary tyrosinaemia type 1 is a rare but serious metabolic disorder with an autosomal recessive mode of inheritance. We describe the prenatal diagnosis of an affected fetus performed by DNA‐mutation analysis and a subsequent pregnancy with a healthy child in the same family.

Börjeson-Forssman-Lehmann Syndrome Due to a Novel Plant Homeodomain Zinc Finger Mutation in the PHF6 Gene

The Börjeson-Forssman-Lehmann syndrome is an X-linked mental retardation disorder caused by mutations in the PHF6 gene. The PHF6 gene contains 2 plant homeodomain zinc fingers, suggesting a role for the protein in chromatin remodeling. In this study, the authors report on a Finnish family with a classical Börjeson-Forssman-Lehmann syndrome phenotype caused by a G to T nucleotide substitution at position 266 within exon 4 within the PHF6 gene (c.266G>T). The resulting glycine to valine (p.G89V) change corresponds to a highly conserved residue within the first plant homeodomain zinc finger domain. This is a novel change that adds to the number of plant homeodomain zinc finger mutations identified, such that 23% of all Börjeson-Forssman-Lehmann syndrome mutations lie within this motif. Moreover, it highlights the functional importance of plant homeodomain zinc finger motifs to human disease and more specifically to PHF6 function.

Identification of C12orf4 as a gene for autosomal recessive intellectual disability

Intellectual disability (ID) is a major health problem in our society. Genetic causes of ID remain unknown because of its vast heterogeneity. Here we report two Finnish families and one Dutch family with affected individuals presenting with mild to moderate ID, neuropsychiatric symptoms and delayed speech development. By utilizing whole exome sequencing (WES), we identified a founder missense variant c.983T>C (p.Leu328Pro) in seven affected individuals from two Finnish consanguineous families and a deletion c.799_1034‐429delinsTTATGA (p.Gln267fs) in one affected individual from a consanguineous Dutch family in the C12orf4 gene on chromosome 12. Both the variants co‐segregated in the respective families as an autosomal recessive trait. Screening of the p.Leu328Pro variant showed enrichment in the North Eastern sub‐isolate of Finland among anonymous local blood donors with a carrier frequency of 1:53, similar to other disease mutations with a founder effect in that region. To date, only one Arab family with a three affected individuals with a frameshift insertion variant in C12orf4 has been reported. In summary, we expand and establish the clinical and mutational spectrum of C12orf4 variants. Our findings implicate C12orf4 as a causative gene for autosomal recessive ID.

Report of interstitial 22q13.1q13.2 microduplication in two siblings with distinctive dysmorphic features, heart defect and mental retardation

We present two siblings (a boy and a girl) with a submicroscopic 4 Mb duplication at 22q13.1q13.2. Both children manifested infantile hypotonia and delayed motor milestones, congenital heart defect, growth deficiency, and strikingly similar and distinctive craniofacial dysmorphism including brachycephaly, blepharophimosis, short broad-based nose and wide mouth with thin upper lip. The boy had also a submucous cleft palate. Both had fair skin and hair compared with their parents. Both had moderate mental retardation associated with a short attention span. A 4-Mb interstitial duplication at 22q13.1q13.2 was detected by whole genome microarray comparative genomic hybridisation (array CGH) in both children. The duplication was confirmed by fluorescence in situ hybridisation (FISH) analysis. Their parents had normal array CGH results. FISH analysis revealed that the father was a carrier of a balanced interchromosomal submicroscopic insertion of 22q13 into chromosome 11q23, explaining the unbalanced aberration detected in both children. This report narrows down the critical region at 22q13.1q13.2, which is associated with mental retardation, pre- and post-natal growth retardation, hippocampal malformation, psychiatric symptoms such as short attention span and facial dysmorphism including hypertelorism, epicanthal folds and low set/abnormal ears.

Prenatal Diagnosis of Single Gene Disorders in Northern Finland

Prenatal diagnosis of single gene disorders is challenging because of the multidisciplinary diagnostic approach and the genetic counselling needed. Out of 3100 pregnancies of northern Finnish women studied for early detection of congenital defects or genetic disease, 110 were at risk for and 15 were unexpectedly found to carry a fetus with a single gene disorder. Seven of the 21 diseases studied were typically representative of inherited diseases in northern Finland. The diagnostic approaches used included fetal ultrasonography, analysis of the components of the amniotic fluid, fetal enzyme analysis, fetal karyotyping for the fragile X syndrome, and fetal DNA analysis. Altogether, 50 pregnancies were found to be affected; 11 of these were continued because the diagnosis was too late for termination, and seven because the parents wanted it. Prenatal diagnosis of severe hereditary disease can be enhanced by improving the detection of pregnancies at risk, and by developing methods for earlier fetal diagnosis.