Tommaso Rizzuti

Loss‐of‐Function FANCL Mutations Associate with Severe Fanconi Anemia Overlapping the VACTERL Association

The diagnosis of VACTERL syndrome can be elusive, especially in the prenatal life, due to the presence of malformations that overlap those present in other genetic conditions, including the Fanconi anemia (FA). We report on three VACTERL cases within two families, where the two who arrived to be born died shortly after birth due to severe organs’ malformations. The suspicion of VACTERL association was based on prenatal ultrasound assessment and postnatal features. Subsequent chromosome breakage analysis suggested the diagnosis of FA. Finally, by next‐generation sequencing based on the analysis of the exome in one family and of a panel of Fanconi genes in the second one, we identified novel FANCL truncating mutations in both families. We used ectopic expression of wild‐type FANCL to functionally correct the cellular FA phenotype for both mutations. Our study emphasizes that the diagnosis of FA should be considered when VACTERL association is suspected. Furthermore, we show that loss‐of‐function mutations in FANCL result in a severe clinical phenotype characterized by early postnatal death.

Nasopharyngeal teratoma and diaphragmatic hernia: a non random association?

We describe a female affected by diaphragmatic hernia and nasopharyngeal teratoma. The case is compared with one already reported and possible diagnoses discussed. These cases appear to represent a new syndrome.

Prenatal diagnosis of genetic syndromes may be facilitated by serendipitous findings at fetal blood sampling.

Two women without a specific risk had fetuses with multiple malformations diagnosed by ultrasound; extensive biochemical investigations on fetal blood revealed clues which would have allowed the correct diagnosis of a genetic condition: Pallister–Killian syndrome in one with increased fetal LDH, and Smith–Lemli–Opitz type II syndrome in the other with low fetal cholesterolaemia. When compared with chorionic villus sampling and amniocentesis, rapid karyotyping in women with multiple fetal malformations by fetal blood sampling allows the collection of additional data which may lead to the diagnosis of specific genetic syndromes.

Changes in Whole-Body Oxygen Consumption and Skeletal Muscle Mitochondria During Linezolid-Induced Lactic Acidosis.

Objective: To better clarify the pathogenesis of linezolid-induced lactic acidosis. Design: Case report. Setting: ICU. Patient: A 64-year-old man who died with linezolid-induced lactic acidosis. Interventions: Skeletal muscle was sampled at autopsy to study mitochondrial function. Measurements and Main Results: Lactic acidosis developed during continuous infusion of linezolid while oxygen consumption and oxygen extraction were diminishing from 172 to 52 mL/min/m2 and from 0.27 to 0.10, respectively. Activities of skeletal muscle respiratory chain complexes I, III, and IV, encoded by nuclear and mitochondrial DNA, were abnormally low, whereas activity of complex II, entirely encoded by nuclear DNA, was not. Protein studies confirmed stoichiometric imbalance between mitochondrial (cytochrome c oxidase subunits 1 and 2) and nuclear (succinate dehydrogenase A) DNA–encoded respiratory chain subunits. These findings were not explained by defects in mitochondrial DNA or transcription. There were no compensatory mitochondrial biogenesis (no induction of nuclear respiratory factor 1 and mitochondrial transcript factor A) or adaptive unfolded protein response (reduced concentration of heat shock proteins 60 and 70). Conclusions: Linezolid-induced lactic acidosis is associated with diminished global oxygen consumption and extraction. These changes reflect selective inhibition of mitochondrial protein synthesis (probably translation) with secondary mitonuclear imbalance. One novel aspect of linezolid toxicity that needs to be confirmed is blunting of reactive mitochondrial biogenesis and unfolded protein response.

Prenatal diagnosis of Simpson-Golabi-Behmel syndrome.

Simpson–Golabi–Behmel syndrome (SGBS) is an overgrowth syndrome and it is usually diagnosed postnatally, on the basis of phenotype. Prenatal ultrasonography may show fetal alterations, but they are not pathognomonic and most of them are frequently detectable only from the 20th week of gestation. Nevertheless, early diagnosis is important to avoid neonatal complications and make timely and informed decisions about the pregnancy. We report on four fetuses from two unrelated families, in whom the application of whole exome sequencing and array‐CGH allowed the identification of GPC3 alterations causing SGBS. The careful follow up of pregnancies and more sophisticated analysis of ultrasound findings led to the identification of early prenatal alterations, which will improve the antenatal diagnosis of SGBS.

Cumming syndrome with heterotaxia, campomelia and absent uterus/fallopian tubes

We have read with interest the article by Ming et al. [1997], regarding the presence of laterality defects in a fetus with Cumming syndrome. In 1986 Cumming et al. described a stillborn male infant, born at 27 weeks of gestation, who had bowed limbs,marked cervical lymphocele, polycystic dysplasia of kidneys, pancreas and liver, and polysplenia. Subsequently, Urioste et al. [1991] andPerez delRio et al. [1999] reported four cases with similar findings. We observed a 46,XX fetus with ascites, campomelia, multicystic dysplastic kidneys, polysplenia, laterality defects of heart and lungs, findings reported previously by Ming et al. [1997]. In our case, tubes and uterus were absent—a finding not described before. The patient was the first-born of healthy, non-consanguineous Caucasian parents. Family history was unremarkable. At 21st week, oligohydramnios and skeletal dysplasia were suspected. Ultrasound examination at 22nd week showed thoracichypoplasia,ascites,mildhyperchogenickidneys,oligohydramnios, shortness, and bowing of the lower limbs. Femur and tibia lengths were, respectively, 26.8 mm (<5th centile) and 27.3 mm (5th centile). The fetal stomach was not visualized and there was a single umbilical artery. Fetal blood chromosomes were normal (46,XX). The couplewas informedabout theprognosis andunderwent a termination at 23rd week. This female infant weighed 581 g (normal 400–550 g), and was 25.5 cm long (normal 25–32 cm). The palpebral fissures were oedematous, nasal bridge was flat, mouth was small and

Prenatal and postnatal findings in five cases of Fryns syndrome

It is characterized by a variety of congenitalanomalies, such as diaphragmatic hernia, facial dysmorphisms(coarse face, hypertelorism, broad and flat nasal bridge withthick nasal tip, long philtrum, tented upper lip, wide mouth,micrognathia, low-set and poorly formed ears), distal digitalhypoplasia, and others (cerebral, ocular, cardiovascular,genitourinary).

Delineating the Mosaic Trisomy 15 Phenotype Using a Serendipitous Mechanism as a Clue

Parental balanced translocation is one of the traditional indications for invasive prenatal diagnosis. Usually, the diagnostic process is straightforward. Sometimes, however, results are not entirely clear and may reveal unexpected biological processes. We performed chorionic villi sampling for a paternal 8;15 reciprocal translocation in the sixth pregnancy of a Caucasian woman. Cytogenetic analysis of chorionic villi, after both short- and long-term cultures, revealed the presence of the same rearrangement found in the father as well as a trisomy 15. Surprisingly, the trisomy, which was initially expected to derive from aberrant segregation during paternal meiosis, resulted instead from maternal nondisjunction. Although a sonogram of the fetus appeared to be normal, follow-up amniocentesis demonstrated a low-level mosaic trisomy 15 in cells extracted from the amniotic fluid, while 10% of cells from fetal tissues sampled after termination of the pregnancy were also found to be trisomic. Fetal autopsy showed dysmorphic features, confirming the diagnosis of mosaic trisomy 15 and enabled deeper insight into the prenatal phenotype of this rare condition.

Prenatal upper-limb mesomelia and 2q31.1 microdeletions affecting the regulatory genome

To the Editor: We read with great interest the article “Noncoding Copy-Number Variations Are Associated With Congenital Limb Malformation,” by Flöttmann et al.1 Their work represents the first large-scale study assessing copynumber variants (CNVs) in individuals with limb defects, showing that the majority of the pathogenic variants are noncoding CNVs affecting regulatory elements. Besides describing previously known disease-associated CNVs, the authors studied in detail four novel candidate loci, which caught our attention and encouraged us to share our experience with a prenatal case. The patient was a male fetus—the first pregnancy of unrelated healthy parents with unremarkable family history. After an uneventful first trimester, ultrasound at 21 weeks and 1 day showed limb abnormalities: the radius and ulna were 19.8 and 15.1 mm, respectively (significantly less than the second percentile), with bowing of the radii and ulnar deviation of the hands. Occipital frontal circumference, humerus, femur, tibia, fibula, and foot measurements were within the normal range, and no other malformations were detected. Array comparative genomic hybridization (SurePrint G3 Human CGH Microarray Kit; 60 K) performed on a chorionic villus sample showed a 998-kb microdeletion on 2q31.1 (175,904,189–176,902,313; GRch37/hg19), de novo. Higherresolution array comparative genomic hybridization (180 K) confirmed this finding and detected a second, more telomeric de novo microdeletion of 153 kb on 2q31.1 (177,146,631– 177,299,841). The parents elected for termination. Fetal autopsy at 22 weeks showed length, weight, and occipital frontal circumference consistent with age, and confirmed upperlimb mesomelia: the humerus length was 4.4 cm (50th percentile), and the ulna length was 2.4 cm (less than the 2nd percentile). Diffuse cutaneous edema and minor facial anomalies were noted in the absence of other congenital malformations (Supplementary Figure S1A online). Mesomelic involvement confined to the upper limbs was confirmed on skeletal survey (Supplementary Figure S1B). The second pregnancy of the couple resulted in a healthy baby with normal chromosomes. In humans, segmental identity and patterning along the body axis is specified by the spatiotemporal expression of four gene clusters that encode the evolutionarily conserved HOX genes: HOXA–D. The HOXD cluster on chromosome 2q31.1 is surrounded by two topologically associated domains (TADs) containing long-range enhancers and regulatory elements for the HOXD genes.2 Mouse models showed that this cluster is regulated in a manner such that gene expression along the anterior–posterior body axis correlates with their physical order on 2q31.1—a phenomenon known as spatial and temporal collinearity.2 Regulatory elements within the telomeric TAD are responsible for early proximal limb development (arm and forearm), while those within the centromeric TAD regulate later distal limb development (hands).2 The chromosomal microarray results in our patient excluded the involvement of any of the genes in the HOXD cluster, but rather the deletions impacted both TADs (Supplementary Figure S1C online). The 998-kb microdeletion involves ATF2, ATP5G3 and the limb-expressed gene KIAA1715 (Lunapark; Lnp), as well as a 40-kb enhancer that controls both KIAA1715 and genes within the HOXD cluster itself. Notably, the second, 153-kb microdeletion involves the telomeric TAD. It also disrupts MTX2, which encodes a mitochondrial membrane protein not expressed in the limb bud, and thus unlikely to contribute to the phenotype in our patient. Of note, a well-characterized mouse mutant, Ulnaless, exhibits markedly reduced ulna and radius, and small tibia and fibula as a result of a balanced paracentric inversion, whose centromeric breakpoint resides in KIAA1715 and telomeric breakpoint is positioned 770 kb downstream from MTX2.2 In contrast to the Ulnaless mouse mutant, the skeletal defect in our patient is confined to the upper limbs, inconsistent with a more generalized skeletal dysplasia. We therefore searched the literature for similar structural variants and mesomelic dysplasia (a heterogeneous group of skeletal dysplasias) in humans. Interestingly, whereas both KIAA1715 and MTX2 are duplicated in Kantaputra-type mesomelic dysplasia (OMIM 156232) and variably disrupted in patients with mesomelia, the sparing of the lower limbs distinguishes our patient from Kantaputra-type mesomelic dysplasia. Indeed, our patient more closely resembles those described by other authors, with normal height and sparing of the lower limbs (Supplementary Table S1 online). One family was found to have a balanced translocation t(2;8) with the chromosome 2 breakpoint located 21 kb from MTX2.3 Another patient had a duplication involving only MTX2, but not KIAA1715, and exclusive or predominant involvement of the upper limbs.4 Although differences in resolution of the various cytogenetic tests used make it difficult to compare breakpoints, these cases, together with ours and the new findings by Flöttmann et al.,1 point to deregulation of the spatiotemporal expression of the HOXD cluster as the underlying cause, thus representing one of only a few conditions so far ascribed to the disruption of TADs.

Fetal death associated with diffuse mesangial sclerosis combined with bilateral multicystic kidney

Abstract Prenatal diagnosis of kidney disease has been greatly increased due to the improvement of antenatal ultrasound techniques, even if a level of uncertainty in differential diagnosis still leads to difficulties in the prediction of fetal outcome and in the counseling of parents. We have followed with ultrasound a 36-year-old secundigravida, referred for fetal renal ultrasound evaluation because of enlarged bilateral cystic kidneys and anhydramnios. Karyotype was normal (46,XY). On the 21st week, the kidneys were slightly enlarged with normal morphology. Beginning with the 28th week, repeated scans showed a reduction of amniotic fluid and an increasing number of renal cysts. Pregnancy was followed to the 37th week when fetal death occurred without warning. Postmortem gross examination showed bilateral enlarged kidneys and histology-defined diffuse mesangial sclerosis with poorly developed cortex and renal dysplasia with multiple cysts. This is an unusual case of multiple renal congenital abnormalities difficult to interpret and which made necessary a multidisciplinary approach due to its complexity. Nevertheless, etiology was not defined. The broad spectrum of antenatal manifestation of prenatally onset kidney diseases requires the identification of more prognostic signs to define fetal outcome and improve parental guidance.