Anna Hammarsjö

22q11.2 microduplication in two patients with bladder exstrophy and hearing impairment

Bladder exstrophy is a congenital malformation of the bladder and urethra. The genetic basis of this malformation is unknown however it is well known that chromosomal aberrations can lead to defects in organ development. A few bladder exstrophy patients have been described to carry chromosomal aberrations. Chromosomal rearrangements of 22q11.2 are implicated in several genomic disorders i.e. DiGeorge/velocardiofacial- and cat-eye syndrome. Deletions within this chromosomal region are relatively common while duplications of 22q11.2 are much less frequently observed. An increasing number of reports of microduplications of this region describe a highly variable phenotype. We have performed array-CGH analysis of 36 Swedish bladder exstrophy patients. The analysis revealed a similar and approximately 3 Mb duplication, consistent with the recently described 22q11.2 microduplication syndrome, in two unrelated cases with bladder exstrophy and hearing impairment. This finding was confirmed by multiplex ligation-dependent probe amplification (MLPA) and FISH analysis. Subsequent MLPA analysis of this chromosomal region in 33 bladder exstrophy patients did not reveal any deletion/duplication within this region. MLPA analysis of 171 anonymous control individuals revealed one individual carrying this microduplication. This is the first report of 22q11.2 microduplication associated with bladder exstrophy and hearing impairment. Furthermore the finding of one carrier among a cohort of normal controls further highlights the variable phenotype linked to this microduplication syndrome.

A novel phenotype in N-glycosylation disorders: Gillessen-Kaesbach-Nishimura skeletal dysplasia due to pathogenic variants in ALG9.

A rare lethal autosomal recessive syndrome with skeletal dysplasia, polycystic kidneys and multiple malformations was first described by Gillessen-Kaesbach et al and subsequently by Nishimura et al. The skeletal features uniformly comprise a round pelvis, mesomelic shortening of the upper limbs and defective ossification of the cervical spine. We studied two unrelated families including three affected fetuses with Gillessen-Kaesbach–Nishimura syndrome using whole-exome and Sanger sequencing, comparative genome hybridization and homozygosity mapping. All affected patients were shown to have a novel homozygous splice variant NM_024740.2: c.1173+2T>A in the ALG9 gene, encoding alpha-1,2-mannosyltransferase, involved in the formation of the lipid-linked oligosaccharide precursor of N-glycosylation. RNA analysis demonstrated skipping of exon 10, leading to shorter RNA. Mass spectrometric analysis showed an increase in monoglycosylated transferrin as compared with control tissues, confirming that this is a congenital disorder of glycosylation (CDG). Only three liveborn children with ALG9-CDG have been previously reported, all with missense variants. All three suffered from intellectual disability, muscular hypotonia, microcephaly and renal cysts, but none had skeletal dysplasia. Our study shows that some pathogenic variants in ALG9 can present as a lethal skeletal dysplasia with visceral malformations as the most severe phenotype. The skeletal features overlap with that previously reported for ALG3- and ALG12-CDG, suggesting that this subset of glycosylation disorders constitutes a new diagnostic group of skeletal dysplasias.

GSTM1 Gene Expression Correlates to Leiomyoma Volume Regression in Response to Mifepristone Treatment

Progesterone receptor modulators, such as mifepristone are useful and well tolerated in reducing leiomyoma volume although with large individual variation. The objective of this study was to investigate the molecular basis for the observed leiomyoma volume reduction, in response to mifepristone treatment and explore a possible molecular marker for the selective usage of mifepristone in leiomyoma patients. Premenopausal women (N = 14) were treated with mifepristone 50 mg, every other day for 12 weeks prior to surgery. Women were arbitrarily sub-grouped as good (N = 4), poor (N = 4) responders to treatment or intermediate respondents (N = 3). Total RNA was extracted from leiomyoma tissue, after surgical removal of the tumour and the differential expression of genes were analysed by microarray. The results were analysed using Ingenuity Pathway Analysis software. The glutathione pathway was the most significantly altered canonical pathway in which the glutathione-s transferase mu 1 (GSTM1) gene was significantly over expressed (+8.03 folds) among the good responders compared to non responders. This was further confirmed by Real time PCR (p = 0.024). Correlation of immunoreactive scores (IRS) for GSTM1 accumulation in leiomyoma tissue was seen with base line volume change of leiomyoma R = −0.8 (p = 0.011). Furthermore the accumulation of protein GSTM1 analysed by Western Blot correlated significantly with the percentual leiomyoma volume change R = −0.82 (p = 0.004). Deletion of the GSTM1 gene in leiomyoma biopsies was found in 50% of the mifepristone treated cases, with lower presence of the GSTM1 protein. The findings support a significant role for GSTM1 in leiomyoma volume reduction induced by mifepristone and explain the observed individual variation in this response. Furthermore the finding could be useful to further explore GSTM1 as a biomarker for tailoring medical treatment of uterine leiomyomas for optimizing the response to treatment. Clinical Trials identifier www.clinicaltrials.gov: NCT00579475, Protocol date: November 2004. http://clinicaltrials.gov/ct2/show/NCT00579475

Extending the phenotype of BMPER-related skeletal dysplasias to ischiospinal dysostosis

Ischiospinal dysostosis (ISD) is a polytopic dysostosis characterized by ischial hypoplasia, multiple segmental anomalies of the cervicothoracic spine, hypoplasia of the lumbrosacral spine and occasionally associated with nephroblastomatosis. ISD is similar to, but milder than the lethal/semilethal condition termed diaphanospondylodysostosis (DSD), which is associated with homozygous or compound heterozygous mutations of bone morphogenetic protein-binding endothelial regulator protein (BMPER) gene. Here we report for the first time biallelic BMPER mutations in two patients with ISD, neither of whom had renal abnormalities. Our data supports and further extends the phenotypic variability of BMPER-related skeletal disorders.

Expanding the Clinical Spectrum of Phenotypes Caused by Pathogenic Variants in PLOD2

Osteogenesis imperfecta (OI) is a strikingly heterogeneous group of disorders with a broad range of phenotypic variations. It is also one of the differential diagnoses in bent bone dysplasias along with campomelic dysplasia and thanatophoric dysplasia and can usually be distinguished by decreased bone mineralization and bone fractures. Bent bone dysplasias also include syndromes such as kyphomelic dysplasia (MIM:211350) and mesomelic dysplasia Kozlowski‐Reardon (MIM249710), both of which have been under debate regarding whether or not they are a real entity or simply a phenotypic manifestation of another dysplasia including OI. Bruck syndrome type 2 (BRKS2; MIM:609220) is a rare form of autosomal recessive OI caused by biallelic PLOD2 variants and is associated with congenital joint contractures with pterygia. In this report, we present six patients from four families with novel PLOD2 variants. All cases had multiple fractures. Other features ranged from prenatal lethal severe angulation of the long bones as in kyphomelic dysplasia and mesomelic dysplasia Kozlowski‐Reardon through classical Bruck syndrome to moderate OI with normal joints. Two siblings with a kyphomelic dysplasia‐like phenotype who were stillborn had compound heterozygous variants in PLOD2 (p.Asp585Val and p.Ser166*). One infant who succumbed at age 4 months had a bent bone phenotype phenotypically like skeletal dysplasia Kozlowski‐Reardon (with mesomelic shortening, camptodactyly, retrognathia, cleft palate, skin dimples, but also with fractures). He was homozygous for the nonsense variant (p.Trp561*). Two siblings had various degrees of Bruck syndrome caused by the homozygous missense variant, p.His687Arg. Furthermore a boy with a clinical presentation of moderate OI had a possibly pathogenic homozygous variant p.Trp588Cys. Our experience of six patients with biallelic pathogenic variants in PLOD2 expands the phenotypic spectrum in the PLOD2‐related phenotypes.

Autosomal recessive mutations in the COL2A1 gene cause severe spondyloepiphyseal dysplasia

To the Editor, Spondyloepiphyseal dysplasia congenita (SEDC, MIM 183900) is a skeletal dysplasia caused by heterozygous mutations in COL2A1, the gene encoding α1-chains of type II collagen (1). Affected individuals are disproportionately short with spinal deformities, cleft palate, myopia and hearing impairment. Skeletal surveys show platyspondyly, retarded ossification of pubic bones, epiphyseal and occasionally metaphyseal abnormalities (1). Here we report on a patient with severe SEDC and homozygosity for a novel COL2A1 mutation: p.Arg437Trp. The proband is the first child of second cousins from India with no familial history of skeletal dysplasia. He

22q11.2 Microduplication in Two Patients with Bladder Exstrophy and Hearing Impairment

Purpose Bladder exstrophy is a congenital malformation of the bladder occurring in about 1 in 35 000 newborns. A few cases of bladder exstrophy patients have been described to carry chromosomal aberrations and in addition the recurrence risk in higher among relatives of bladder exstrophy cases than in the general population. This speaks in favour of a genetic contribution to the pathogenesis of bladder exstrophy. Material and Methods We have performed array-CGH analysis of 36 Swedish bladder exstrophy patients in order to look for submicroscopic chromosomal aberrations. Results The analysis revealed a 22q11.21 microduplication in two unrelated cases with bladder exstrophy and hearing impairment consistent with the recently described 22q11.2 microduplication syndrome. This finding was confirmed by multiplex ligation-dependent probe amplification (MLPA) and FISH analysis. Subsequent MLPA analysis of this chromosomal region in 33 bladder exstrophy patients did not reveal any deletion/duplication within this region. MLPA analysis of 171 anonymous control individuals revealed one individual carrying this microduplication. Conclusions Chromosomal rearrangements of 22q11.2 are implicated in several genomic disorders i.e. DiGeorge/velocardiofacial- and cat-eye syndrome. Deletions within this chromosomal region are relatively common while duplications of 22q11.2 are much less frequently observed. An increasing number of reports of microduplications of this region describe a highly variable phenotype. This is the first report of 22q11.2 microduplication associated with bladder exstrophy and hearing impairment. The finding of one carrier among a cohort of normal controls further highlights the variable phenotype linked to this microduplication syndrome.

Genotype-Phenotype Correlation of PLOD2 Skeletal Dysplasias Using Structural Information: REPLY LETTER TO THE EDITOR

To the Editor: Guo and colleagues havedetermined the2Å crystal structureof the lysyl hydroxylase (LH) domain (amino acids 680–895 corresponding to human PLOD2 amino acids 548–758; Fig. 1) of L230, an enzyme of Acanthamoeba polyphaga mimivirus (APMV). They show that this domain confers metastatic potential to cancer cells. Human cells use the same pathways in constitutional disorders and in cancer, thus DNA and protein studies in both fields may assist our understanding of the molecular pathophysiology of both congenital syndromes and cancer. Guo and colleagues’ study shows that LH forms homodimers through hydrophobic contacts and that dimerization enables LH activity on natural collagen substrates. They also point out amino acids important for the hydrophobic contacts in the dimer and show that the binding site for Fe2þ is composed of the highly conserved His825, Asp827, and His877 in L230 (corresponding to human PLOD2 amino acid positions His687, Asp689, and His739). Our recent study using massive parallel sequencing recently disclosed that the PLOD2-skeletal dysplasia family contains lethal phenotypes, previously classified as kyphomelic dysplasia and a mesomelic dysplasia KozlowskiReardon-like entity, and a milder phenotype of moderate osteogenesis imperfecta (OI), in addition to the classic phenotype of Brucks syndrome. To date, there is no known genotypephenotype correlation in the PLOD2-family of skeletal dysplasias. There are three reported families with lethally affected individuals, including family 1 and2 inour study andone familywith three affected siblings homozygous for a missense mutation, p. (Thr629Ile). Threonine in position 629 is highly conserved down to L230 of APMV and is located in the LH domain. The homozygous nonsense mutation p.(Trp561 ) in our family 2 is predicted to remove the entire LH domain, whereas compound heterozygous mutations (in our family 1) p.(Ser166 ) and p. (Asp585Val) also are predicted to lead to loss of LH domain and alteration of the highly evolutionarily conserved amino acid 585. The homozygous missense mutation, p.His687Arg, found in our family 3 with two siblings with classical Brucks syndrome was located inoneof the aminoacids shownbyGuoandcolleagues to bind Fe2þ. The homozygous variant found in ourmildest affected patient with phenotype of moderate OI (but no classical features of Brucks syndrome), p.(Trp588Cys), was in a position that was within the LH domain but not conserved in L230 (which had a Serine in that position). We speculate that cysteine may create novel disulphide bonds that might change the protein structure. The protein crystallography of the highly conserved L230domain homologous to the LH domain in PLOD2 by Guo and colleagues combined with information from studies mapping human mutations indicates that there might be a genotypephenotype correlation in PLOD2-related skeletal dysplasias; ie, damage of structurally more important amino acids leads to more severe phenotypes. The number of currently reported individuals with PLOD2 mutations is still low (n1⁄4 21) and, therefore, structural correlations must be interpreted with care. However, further genomic studies will make it possible to identify individuals with both milder and more severe PLOD2-related phenotypes. Basic scientists working hand in hand with clinicians, eg, translational studies, are of key importance for identifying the basic molecular mechanisms used by human cells. Better definition of the human PLOD1-3 structure and the position and role of their mutations as well as functional studies of the mutated protein may lead to better understanding of enzyme function, contribute to improved interpretation of rare missense variants, and lead to key insights needed for the development of possible LH activity-modulating molecules.

Alu-Alu mediated intragenic duplications in IFT81 and MATN3 are associated with skeletal dysplasias

Skeletal dysplasias are a diverse group of rare Mendelian disorders with clinical and genetic heterogeneity. Here, we used targeted copy number variant (CNV) screening and identified intragenic exonic duplications, formed through Alu‐Alu fusion events, in two individuals with skeletal dysplasia and negative exome sequencing results. First, we detected a homozygous tandem duplication of exon 9 and 10 in IFT81 in a boy with Jeune syndrome, or short‐rib thoracic dysplasia (SRTD) (MIM# 208500). Western blot analysis did not detect any wild‐type IFT81 protein in fibroblasts from the patient with the IFT81 duplication, but only a shorter isoform of IFT81 that was also present in the normal control samples. Complementary zebrafish studies suggested that loss of full‐length IFT81 protein but expression of a shorter form of IFT81 protein affects the phenotype while being compatible with life. Second, a de novo tandem duplication of exons 2 to 5 in MATN3 was identified in a girl with multiple epiphyseal dysplasia (MED) type 5 (MIM# 607078). Our data highlights the importance of detection and careful characterization of intragenic duplication CNVs, presenting them as a novel and very rare genetic mechanism in IFT81‐related Jeune syndrome and MATN3‐related MED.