Ricarda Flöttmann

Effective diagnosis of genetic disease by computational phenotype analysis of the disease-associated genome

Patients with genetic disease of unknown causes can be rapidly diagnosed by bioinformatic analysis of disease-associated DNA sequences and phenotype. Efficient Diagnosis of Genetic Disease We know which genes are mutated in almost 3000 inherited human diseases and have good descriptions of how these mutations affect the human phenotype. Now, Zemojtel et al. have coupled this knowledge with rapid sequencing of these genes in a group of 40 patients with undiagnosed genetic diseases. Bioinformatic matching of the patients’ clinical characteristics and their disease gene sequences to databases of current genetic and phenotype knowledge enabled the authors to successfully diagnose almost 30% of the patients. The process required only about 2 hours of a geneticists’ time. Zemojtel et al. have made their tools available to the community, enabling a fast straightforward process by which clinicians and patients can easily identify the genetic basis of inherited disease in certain people. Less than half of patients with suspected genetic disease receive a molecular diagnosis. We have therefore integrated next-generation sequencing (NGS), bioinformatics, and clinical data into an effective diagnostic workflow. We used variants in the 2741 established Mendelian disease genes [the disease-associated genome (DAG)] to develop a targeted enrichment DAG panel (7.1 Mb), which achieves a coverage of 20-fold or better for 98% of bases. Furthermore, we established a computational method [Phenotypic Interpretation of eXomes (PhenIX)] that evaluated and ranked variants based on pathogenicity and semantic similarity of patients’ phenotype described by Human Phenotype Ontology (HPO) terms to those of 3991 Mendelian diseases. In computer simulations, ranking genes based on the variant score put the true gene in first place less than 5% of the time; PhenIX placed the correct gene in first place more than 86% of the time. In a retrospective test of PhenIX on 52 patients with previously identified mutations and known diagnoses, the correct gene achieved a mean rank of 2.1. In a prospective study on 40 individuals without a diagnosis, PhenIX analysis enabled a diagnosis in 11 cases (28%, at a mean rank of 2.4). Thus, the NGS of the DAG followed by phenotype-driven bioinformatic analysis allows quick and effective differential diagnostics in medical genetics.

Evaluating the performance of the breast cancer genetic risk models BOADICEA, IBIS, BRCAPRO and Claus for predicting BRCA1/2 mutation carrier probabilities: a study based on 7352 families from the German Hereditary Breast and Ovarian Cancer Consortium

Background Risk prediction models are widely used in clinical genetic counselling. Despite their frequent use, the genetic risk models BOADICEA, BRCAPRO, IBIS and extended Claus model (eCLAUS), used to estimate BRCA1/2 mutation carrier probabilities, have never been comparatively evaluated in a large sample from central Europe. Additionally, a novel version of BOADICEA that incorporates tumour pathology information has not yet been validated. Patients and methods Using data from 7352 German families we estimated BRCA1/2 carrier probabilities under each model and compared their discrimination and calibration. The incremental value of using pathology information in BOADICEA was assessed in a subsample of 4928 pedigrees with available data on breast tumour molecular markers oestrogen receptor, progesterone receptor and human epidermal growth factor 2. Results BRCAPRO (area under receiver operating characteristic curve (AUC)=0.80 (95% CI 0.78 to 0.81)) and BOADICEA (AUC=0.79 (0.78–0.80)), had significantly higher diagnostic accuracy than IBIS and eCLAUS (p<0.001). The AUC increased when pathology information was used in BOADICEA: AUC=0.81 (95% CI 0.80 to 0.83, p<0.001). At carrier thresholds of 10% and 15%, the net reclassification index was +3.9% and +5.4%, respectively, when pathology was included in the model. Overall, calibration was best for BOADICEA and worst for eCLAUS. With eCLAUS, twice as many mutation carriers were predicted than observed. Conclusions Our results support the use of BRCAPRO and BOADICEA for decision making regarding genetic testing for BRCA1/2 mutations. However, model calibration has to be improved for this population. eCLAUS should not be used for estimating mutation carrier probabilities in clinical settings. Whenever possible, breast tumour molecular marker information should be taken into account.

Microduplications encompassing the Sonic hedgehog limb enhancer ZRS are associated with Haas‐type polysyndactyly and Laurin‐Sandrow syndrome

Laurin‐Sandrow syndrome (LSS) is a rare autosomal dominant disorder characterized by polysyndactyly of hands and/or feet, mirror image duplication of the feet, nasal defects, and loss of identity between fibula and tibia. The genetic basis of LSS is currently unknown. LSS shows phenotypic overlap with Haas‐type polysyndactyly (HTS) regarding the digital phenotype. Here we report on five unrelated families with overlapping microduplications encompassing the Sonic hedgehog (SHH) limb enhancer ZPA regulatory sequence (ZRS) on chromosome 7q36. Clinically, the patients show polysyndactyly phenotypes and various types of lower limb malformations ranging from syndactyly to mirror image polydactyly with duplications of the fibulae. We show that larger duplications of the ZRS region (>80 kb) are associated with HTS, whereas smaller duplications (<80 kb) result in the LSS phenotype. On the basis of our data, the latter can be clearly distinguished from HTS by the presence of mirror image polysyndactyly of the feet with duplication of the fibula. Our results expand the clinical phenotype of the ZRS‐associated syndromes and suggest that smaller duplications (<80 kb) are associated with a more severe phenotype. In addition, we show that these small microduplications within the ZRS region are the underlying genetic cause of Laurin‐Sandrow syndrome.

Exome sequencing and CRISPR/Cas genome editing identify mutations of ZAK as a cause of limb defects in humans and mice

The CRISPR/Cas technology enables targeted genome editing and the rapid generation of transgenic animal models for the study of human genetic disorders. Here we describe an autosomal recessive human disease in two unrelated families characterized by a split-foot defect, nail abnormalities of the hands, and hearing loss, due to mutations disrupting the SAM domain of the protein kinase ZAK. ZAK is a member of the MAPKKK family with no known role in limb development. We show that Zak is expressed in the developing limbs and that a CRISPR/Cas-mediated knockout of the two Zak isoforms is embryonically lethal in mice. In contrast, a deletion of the SAM domain induces a complex hindlimb defect associated with down-regulation of Trp63, a known split-hand/split-foot malformation disease gene. Our results identify ZAK as a key player in mammalian limb patterning and demonstrate the rapid utility of CRISPR/Cas genome editing to assign causality to human mutations in the mouse in <10 wk.

Microdeletions on 6p22.3 are associated with mesomelic dysplasia Savarirayan type

Introduction Mesomelic dysplasias are a group of skeletal disorders characterised by shortness of the middle limb segments (mesomelia). They are divided into 11 different categories. Among those without known molecular basis is mesomelic dysplasia Savarirayan type, characterised by severe shortness of the middle segment of the lower limb. Objective To identify the molecular cause of mesomelic dysplasia Savarirayan type. Methods and results We performed array comparative genomic hybridisation in three unrelated patients with mesomelic dysplasia Savarirayan type and identified 2 Mb overlapping de novo microdeletions on chromosome 6p22.3. The deletions encompass four known genes: MBOAT1, E2F3, CDKAL1 and SOX4. All patients showed mesomelia of the lower limbs with hypoplastic tibiae and fibulae. We identified a fourth patient with intellectual disability and an overlapping slightly larger do novo deletion also encompassing the flanking gene ID4. Given the fact that the fourth patient had no skeletal abnormalities and none of the genes in the deleted interval are known to be associated with abnormalities in skeletal development, other mutational mechanisms than loss of function of the deleted genes have to be considered. Analysis of the genomic region showed that the deletion removes two regulatory boundaries and brings several potential limb enhancers into close proximity of ID4. Thus, the deletion could result in the aberrant activation and misexpression of ID4 in the limb bud, thereby causing the mesomelic dysplasia. Conclusions Our data indicate that the distinct deletion 6p22.3 is associated with mesomelic dysplasia Savarirayan type featuring hypoplastic, triangular-shaped tibiae and abnormally shaped or hypoplastic fibulae.

Duplication of PTHLH causes osteochondroplasia with a combined brachydactyly type E/A1 phenotype with disturbed bone maturation and rhizomelia.

Parathyroid hormone-like hormone (PTHLH, MIM 168470) plays an important role in endochondral bone development and prevents chondrocytes from differentiating. Disease-causing variants and haploinsufficiency of PTHLH are known to cause brachydactyly type E and short stature. So far, three large duplications encompassing several genes including PTHLH associating with enchondromatas and acro-osteolysis have been described in the literature. Here, we report on a three-generation pedigree with short humerus, curved radius, and a specific type of severe brachydactyly with features of types E and A1 but without the enchondromatas and the acro-osteolysis. Microarray-based comparative genomic hybridization (array-CGH) revealed a 70-kb duplication on chromosome 12p11.22 encompassing only PTHLH. Our data extend the phenotypic spectrum associated with copy number variations of PTHLH, and this family is to our knowledge the first description harboring a microduplication encompassing only PTHLH.

Femoral facial syndrome associated with a de novo complex chromosome 2q37 rearrangement.

The femoral facial syndrome (FFS) is a rare congenital anomaly syndrome characterized by bilateral femoral hypoplasia and facial dysmorphism. The etiology of FFS is currently unknown but maternal/gestational diabetes has been proposed as a strong risk factor for syndromic femoral hypoplasia. In affected children born to non‐diabetic mothers, a genetic contribution to FFS is suspected; however, no chromosomal anomalies or gene mutations have been identified so far. Here, we report on a girl with FFS and a de novo complex chromosome rearrangement of terminal chromosome 2q37.2. Radiographs of the pelvis and lower limbs showed bilateral shortening and bowing of the femur and radiographs of hands and feet revealed a brachydactyly type E (BDE). Using high resolution array‐CGH, qPCR, and FISH, we detected a ∼1.9 Mb duplication in the chromosomal region 2q37.2 and a ∼5.4 Mb deletion on chromosome 2q37.3 that were absent in the parents. The duplication contains six genes and the deletion encompasses 68 genes; the latter has previously been shown to cause BDE (through haploinsufficiency for HDAC4) but not femoral hypoplasia. Therefore, we propose that the duplication 2q37.2 could be causative for the femur phenotype. To the best of our knowledge, our report is the first to propose a genetic cause in a case of FFS.

Noncoding copy-number variations are associated with congenital limb malformation

PurposeCopy-number variants (CNVs) are generally interpreted by linking the effects of gene dosage with phenotypes. The clinical interpretation of noncoding CNVs remains challenging. We investigated the percentage of disease-associated CNVs in patients with congenital limb malformations that affect noncoding cis-regulatory sequences versus genes sensitive to gene dosage effects.MethodsWe applied high-resolution copy-number analysis to 340 unrelated individuals with isolated limb malformation. To investigate novel candidate CNVs, we re-engineered human CNVs in mice using clustered regularly interspaced short palindromic repeats (CRISPR)–based genome editing.ResultsOf the individuals studied, 10% harbored CNVs segregating with the phenotype in the affected families. We identified 31 CNVs previously associated with congenital limb malformations and four novel candidate CNVs. Most of the disease-associated CNVs (57%) affected the noncoding cis-regulatory genome, while only 43% included a known disease gene and were likely to result from gene dosage effects. In transgenic mice harboring four novel candidate CNVs, we observed altered gene expression in all cases, indicating that the CNVs had a regulatory effect either by changing the enhancer dosage or altering the topological associating domain architecture of the genome.ConclusionOur findings suggest that CNVs affecting noncoding regulatory elements are a major cause of congenital limb malformations.

A Novel de novo FZD2 Mutation in a Patient with Autosomal Dominant Omodysplasia

We described a heterozygous de novo mutation (G434V) in the frizzled class receptor 2 (FZD2) gene in a patient with distinct facial features including hypertelorism, bilateral cleft lip/palate, short nose with a broad nasal bridge, microretrognathia, and bilateral shortness of the upper limbs, first metacarpal bones, and middle phalanges of the 5th digits. The findings of our patient were compared to an autosomal dominant omodysplasia (OMOD2) family with FZD2 mutation reported in the literature. OMOD2 is a rare skeletal dysplasia and characterized by facial dysmorphism and shortness of the upper extremities and first metacarpal bones. This is the second report which supports the findings of the first family described and points out that heterozygous FZD2 mutations may be disease-causing for OMOD2.

A de novo 1q23.3-q24.2 deletion combined with a GORAB missense mutation causes a distinctive phenotype with cutis laxa

Gerodermia osteodysplastica is a recessive segmental progeroid disorder mainly characterized by wrinkled skin, generalized connective tissue weakness, infantile onset osteoporosis and normal intelligence. Coding mutations in GORAB, localized on chromosome 1q24.2, are the cause of this disease. 1q24 deletions underlie a spectrum of disorders with intellectual disability and ear abnormalities as phenotypic hallmarks. Here we report on an individual from Azerbaijan originating from a non-consanguineous couple showing short stature, cutis laxa, frequent fractures, facial dysmorphism, cup-shaped ears and intellectual disability. Sanger sequencing of GORAB revealed the seemingly homozygous missense mutation p.Ser175Phe. This mutation was detected in a heterozygous state in the clinically unaffected mother, but was absent in the healthy father. We performed copy-number investigations by high-resolution array-CGH and PCR approaches and found an ~6 Mb de novo deletion spanning 1q23.3-q24.2 in the affected boy. This novel combination of genetic defects very well explains the phenotype that goes beyond the usual presentation of gerodermia osteodysplastica. Our data provide new insight into the phenotypic spectrum of 1q23-q25 deletions and shows that the combination with another pathogenic allele can lead to more severe clinical manifestations.