Janice L. B. Byrne

Findings From aCGH in Patients With Congenital Diaphragmatic Hernia (CDH): A Possible Locus for Fryns Syndrome

Congenital diaphragmatic hernia (CDH) is a common and often devastating birth defect that can occur in isolation or as part of a malformation complex. Considerable progress is being made in the identification of genetic causes of CDH. We applied array‐based comparative genomic hybridization (aCGH) of ∼1Mb resolution to 29 CDH patients with prior normal karyotypes who had been recruited into our multi‐site study. One patient, clinically diagnosed with Fryns syndrome, demonstrated a de novo 5Mb deletion at chromosome region 1q41–q42.12 that was confirmed by FISH. Given prior reports of CDH in association with cytogenetic abnormalities in this region, we propose that this represents a locus for Fryns syndrome, a Fryns syndrome phenocopy, or CDH.

Analysis of skeletal dysplasias in the Utah population

The Utah Birth Defect Network (UBDN) collects population‐based data for Utah on births from all resident women. The prevalence of skeletal dysplasias and epidemiologic characteristics/outcomes were evaluated. Cases categorized as a skeletal dysplasia from all live births, stillbirths, and pregnancy terminations (TAB) between 1999 and 2008 were reviewed by three clinical geneticists. After case review, 153 were included for analysis (88% live births, 3% stillborn, 9% TAB), and categorized by groupings defined by molecular, biochemical, and/or radiographic criteria as outlined in the 2010 Nosology and Classification of Genetic Skeletal Disorders. The overall prevalence for skeletal dysplasias was 3.0 per 10,000 births, and 20.0 per 10,000 stillbirths. The most common diagnostic groups were osteogenesis imperfecta (OI; n = 40; 0.79 per 10,000), thanatophoric dysplasia (n = 22; 0.43 per 10,000), achondroplasia (n = 18; 0.35 per 10,000), and cleidocranial dysplasia (n = 6; 0.12 per 10,000). The most common groups based on the 2010 Nosology and Classification of Genetic Skeletal Disorders were the FGFR3 chondrodysplasia group (n = 41; 0.81 per 10,000), the OI/decreased bone density group (n = 40; 0.79 per 10,000), and the type 2 collagen group (n = 10; 0.2 per 10,000). Median age of postnatal diagnosis was 30 days (range 1–2,162). Of those deceased, 88% were prenatally suspected; of those alive 29% prenatally suspected. Median age of death for live born individuals was 1 day (range 1–1,450 days). Previously reported prevalence rates vary, but our data provide a population‐based approach not limited to the perinatal/neonatal period. Understanding the range for survival within each group/diagnosis is beneficial for health care providers when counseling families.

Identification of a prenatal profile of Cornelia de Lange syndrome (CdLS): A review of 53 CdLS pregnancies

Cornelia de Lange Syndrome (CdLS) is a multisystem developmental disorder characterized by growth retardation, cognitive impairment, external and internal structural malformations, and characteristic facial features. Currently, there are no definitive prenatal screening measures that lead to the diagnosis of CdLS. In this study, documented prenatal findings in CdLS syndrome were analyzed towards the development of a prenatal profile predictive of CdLS. We reviewed 53 cases of CdLS (29 previously reported and 24 unreported) in which prenatal observations/findings were available. The review of these cases revealed a pattern of sonographic findings, including obvious associated structural defects, growth restriction, as well as a more subtle, but strikingly characteristic, facial profile, and suggestive of a recognizable prenatal ultrasonographic profile for CdLS. In addition, the maternal serum marker, PAPP‐A, may be reduced and fetal nuchal translucency (NT) may be increased in some pregnancies when measured at an appropriate gestational age. In conclusion, CdLS can be prenatally diagnosed or readily ruled out in a family with a known mutation in a CdLS gene. The characteristic ultrasonographic profile may allow for prenatal diagnosis of CdLS in (1) subsequent pregnancies to a couple with a prior child with CdLS in whom a mutation has not been identified or (2) when there are unexplained pregnancy signs of fetal abnormality, such as oligo‐ or polyhydramnios, a low maternal serum PAPP‐A level and/or increased NT, fetal growth retardation, or structural anomalies consistent with CdLS.

A Family-Based Paradigm to Identify Candidate Chromosomal Regions for Isolated Congenital Diaphragmatic Hernia

Congenital diaphragmatic hernia (CDH) is a developmental defect of the diaphragm that causes high newborn mortality. Isolated or non‐syndromic CDH is considered a multifactorial disease, with strong evidence implicating genetic factors. As low heritability has been reported in isolated CDH, family‐based genetic methods have yet to identify the genetic factors associated with the defect. Using the Utah Population Database, we identified distantly related patients from several extended families with a high incidence of isolated CDH. Using high‐density genotyping, seven patients were analyzed by homozygosity exclusion rare allele mapping (HERAM) and phased haplotype sharing (HapShare), two methods we developed to map shared chromosome regions. Our patient cohort shared three regions not previously associated with CDH, that is, 2q11.2–q12.1, 4p13 and 7q11.2, and two regions previously involved in CDH, that is, 8p23.1 and 15q26.2. The latter regions contain GATA4 and NR2F2, two genes implicated in diaphragm formation in mice. Interestingly, three patients shared the 8p23.1 locus and one of them also harbored the 15q26.2 segment. No coding variants were identified in GATA4 or NR2F2, but a rare shared variant was found in intron 1 of GATA4. This work shows the role of heritability in isolated CDH. Our family‐based strategy uncovers new chromosomal regions possibly associated with disease, and suggests that non‐coding variants of GATA4 and NR2F2 may contribute to the development of isolated CDH. This approach could speed up the discovery of the genes and regulatory elements causing multifactorial diseases, such as isolated CDH.

Fetus with interstitial del(5)(p13.1p14.2) diagnosed postnatally with Cornelia de Lange syndrome

Cornelia de Lange (CdLS), also known as Brachmann de Lange, is a complex disorder that includes characteristic facial features, hirsutism, limb abnormalities, diaphragmatic hernia, preand postnatal growth retardation, and mental retardation. It is estimated to occur in 1 in 10,000 live births [Opitz, 1985]. CdLS is usually diagnosed by postnatal clinical findings; however, prenatal diagnosis has been made by the presence of congenital diaphragmatic hernia, characteristic limb abnormalities, and facial profile, as detected by fetal ultrasound [Urban and Hartung, 2001; Marino et al., 2002]. The gene responsible for CdLS, NIPBL, was recently identified, due in part to the clinical report of this case [Hulinsky et al., 2003] as well as an infant with a balanced de novo translocation (5;13)(p13.1;q12.1) [Krantz et al., 2004]. The proposita was the second child born to a nonconsanguineous Caucasian couple. The mother was referred at 20-week gestation to evaluate abnormal sonographic findings. Targeted ultrasound revealed multiple anomalies including diaphragmatic hernia, single umbilical artery, nuchal thickening, and abnormal upper extremities. Maternal serum screening was normal. The patient had one previous normal pregnancy with delivery at term of a healthy female. The family history was noncontributory. Due to the multiple congenital abnormalities, karyotype analysis by amniocentesis was performed at 20-week gestation. The karyotype was 46,XY,del(5)(p13.1p14.2) at 425 band level, and was confirmed by postnatal chromosome analysis (Fig. 1). Fluorescence in situ hybridization (FISH) analyses usingwhole-chromosome5paint and the cri-du-chat syndrome probe at 5p15.2 demonstrated a simple, interstitial deletion centromeric to the cri-du-chat syndrome deletion breakpoint. Parental karyotypes were normal. The couple was counseled extensively regarding poor prognosis, specifically in the context of a chromosome deletion, oligohydramnios, and diaphragmatic hernia. Follow-up ultrasounds at 26-, 30-, and 34-week gestation showed progressive reduction of amniotic fluid (there was no evidence of premature rupture of membranes) and progressive IUGR. Fetal echocardiogram at 26-week gestation was apparently normal. Precipitous delivery at 36-week gestation resulted in an infant born at home. The parents reported no movement or respiratory effort after delivery. Autopsy demonstrated a male infant with manifestations of Cornelia de Lange and the karyotype was confirmed by tissue biopsy. The facial features were consistent with CdLS, including low hairline, characteristic ‘‘penciled in’’ eyebrows without synophrys, long philtrum, thin lips, upturned nasal tip, and posteriorly rotated and abnormal ears (Fig. 2). Limb abnormalities were also consistent with a diagnosis of CdLS, which included micromelia and oligodactyly of the upper limbs with single forearm bones and short feet. Other external abnormalities consistent with a diagnosis of CdLS included cleft of the soft palate, hirsutism, and hypospadias. Internal abnormalities consistent with a diagnosis of CdLS included left-sided diaphragmatic hernia, and ventricular septal defect. Internal findings of unclear significance included hypoplastic

Germline mosaicism in Cornelia de Lange syndrome.

Cornelia de Lange syndrome (CdLS) is a genetic disorder associated with delayed growth, intellectual disability, limb reduction defects, and characteristic facial features. Germline mosaicism has been a described mechanism for CdLS when there are several affected offspring of apparently unaffected parents. Presently, the recurrence risk for CdLS has been estimated to be as high as 1.5%; however, this figure may be an underrepresentation. We report on the molecularly defined germline mosaicism cases from a large CdLS database, representing the first large case series on germline mosaicism in CdLS. Of the 12 families, eight have been previously described; however, four have not. No one specific gene mutation, either in the NIPBL or the SMC1A gene, was associated with an increased risk for germline mosaicism. Suspected or confirmed cases of germline mosaicism in our database range from a conservative 3.4% up to 5.4% of our total cohort. In conclusion, the potential reproductive recurrence risk due to germline mosiacism should be addressed in prenatal counseling for all families who have had a previously affected pregnancy or child with CdLS.

Group A streptococcal puerperal sepsis: initial characterization of virulence factors in association with clinical parameters

Group A beta-hemolytic streptococcus (GAS) is an uncommon but potentially fatal source of postpartum infection. Pathogenesis in invasive GAS infections has been linked to bacterial virulence factors. In this study, we sought to provide an initial description of potential virulence factors in association with puerperal morbidity by virtue of specific M-protein type antigens. Women with confirmed GAS puerperal infection in the Salt Lake City region were prospectively identified over a 6-year interval (1991-1997). From this cohort, GAS isolates were analyzed with respect to M-serotype and presence of genes encoding the Streptococcal Pyogenic Exotoxins A and B (SPE-A and SPE-B). Bacterial isolates from 18 subjects with GAS puerperal infection underwent M-serotyping and PCR-based genotyping for the speA and speB genes. Among these, 8/18 subjects manifest criteria of severe disease. All 18 isolate strains expressed speB; 6/18 isolates expressed speA. Of the M-serotypes, 8/8 severe disease isolates expressed M-types 1 (N=3) or 28 (N=5). Pulse-field gel electrophoresis did not indicate an outbreak strain among similar isolates. We conclude that in this initial characterization, morbidity among women with GAS puerperal infection is associated with M-types 1 and 28, but not speB genotype.

MORTALITY AND PATHOLOGICAL FINDINGS IN C (OPITZ TRIGONOCEPHALY) SYNDROME

Even as a rare multiple congenital anomalies/mental retardation syndrome, the C-syndrome (CS, or Opitz C-trigonoecephaly syndrome) is, at long last, beginning to attract attention because of its developmental and causal complexity. Also, the possibility that the apparently balanced translocation recently described in an affected Japanese boy may soon provide a molecular/causal insight into this disorder. The manifestations recorded in the previously published patients, those autopsied within recent years, and the unpublished instances in our files suggest that the CS is a heterogeneous genetic disorder, predominantly sporadic but with sufficient familial cases (at times with consanguinity) to allow postulation of an entity due to autosomal dominant mutations with a high rate of germinal mosaicism, or due to both autosomal dominant mutations and an autosomal recessive genocopy. In any event, elucidation of cause and pathogenesis of CS will, in due time, shed light on its developmental pleiotropy, rarity in liveborn infants, prevalence in stillborn fetuses, recurrence risk in humans, and occurrence in other animals (e.g., mice) to further understanding of pathogenesis.

Autosomal Recessive Hypophosphatasia Manifesting in Utero with Long Bone Deformity but Showing Spontaneous Postnatal Improvement

CONTEXT Hypophosphatasia (HPP) is a heritable metabolic disorder of the skeleton that includes variable expressivity conditioned by gene dosage effect and the variety of mutations in the tissue nonspecific alkaline phosphatase (TNSALP) gene. Patient age when skeletal problems first manifest generally predicts the clinical course, with perinatal HPP causing bone disease in utero with postnatal lethality. OBJECTIVE Our objective was to identify TNSALP mutations and characterize the inheritance pattern of a family with clinically variable HPP with one child manifesting in utero with long bone deformity but showing spontaneous prenatal and postnatal improvement. DESIGN TNSALP enzyme and substrate analysis and TNSALP mutation analysis were performed on all family members. PATIENTS A boy with HPP showing long bone deformity that spontaneously improved in utero and after birth is described. His older brother has the childhood form of HPP without findings until after infancy. His parents and twin sister are clinically unaffected. RESULTS Both boys are compound heterozygotes for the same missense mutations in TNSALP, documenting autosomal recessive inheritance for their HPP. The parents each carry one defective allele. CONCLUSIONS The patient is an autosomal recessive case of HPP with prenatal long bone deformity but with spontaneous prenatal and postnatal improvement. Thus, prenatal detection by sonography of bowing of long bones from HPP, even with autosomal recessive inheritance, does not necessarily predict lethality but can represent variable expressivity or the effects of modifiers on the TNSALP defect(s).

Prenatal death in Smith–Lemli–Opitz/RSH syndrome

Smith–Lemli–Opitz (RSH) syndrome is an autosomal recessive disorder involving virtually all organ systems. Severity varies widely, but all degrees of severity are due to the disturbance of the same genetic/biochemical system involving a deficiency of cholesterol [Irons et al., 1993]. The enzyme 7dehydrocholesterol reductase, which is abnormal in RSH syndrome, catalyzes the last step of endogenous cholesterol synthesis [Moebius et al., 1998; Wassif et al., 1998; Waterham et al., 1998]. The gene, DHCR7, which encodes this enzyme, is located on chromosome region 11q13. Some 100 different mutations in the DHCR7 gene have been identified in more than 200 patients with the syndrome [Opitz, 2001]. Cholesterol is a precursor of steroid hormones and bile acids. It is an important component of myelin, mitochondrial and cell membranes [Opitz, 2001; Opitz et al., 2002]. Decreased 7DHCR activity leads to cholesterol deficiency and subsequent accumulation of the precursor molecules 7and 8-dehydrocholesterol. Prenatally, there is a direct correlation between the severity of the phenotype and 7DHC levels in the amniotic fluid. The clinical condition can range from prenatal death with malformations of multiple organ systems to minimal physical effects and only mild behavior problems and mental impairment in children and adults [Nowaczyk et al., 1999, 2001]. Malformations observed in the RSH syndrome include Yshaped 2–3 toe syndactyly, polydactyly, unilobate lungs, renal dysplasia, or agenesis, Hirschsprung disease, complex cardiac malformations, cataracts, central nervous malformations such as microcephaly and agenesis of corpus callosum, oropharyngeal malformations (cleft palate), genital ambiguity in genetic males, and facial abnormalities such as anteverted nostrils, micrognathia, and apparently low-set ears [Smith et al., 1964; Cunniff et al., 1997; Kelley, 2000]. Opitz et al. [2002] described three cases studied pathologically. Case 1 involved a spontaneous miscarriage of an approximately 19-week gestational age female fetus. Ultrasonography had demonstrated an enlarged third ventricle, thickening of the myocardium and oligohydramnios. Autopsy documented a large secundum atrial septal defect, muscular ventricular septal defect, hypertrophy of the left ventricle and enlargement of the right ventricle. The severely hypoplastic lungs were unilobate with an incipient fissure on the right. Hepatomegaly, bilateral renal agenesis with rudimentary ureters, and a bifid uterus were identified. The umbilical cord had three vessels. Case 2 was confirmed by gas chromatography mass-spectrometry of amniotic fluid. The cholesterol content was 6.2 mg/ml; 7and 8-dehydrocholesterol levels were 2.2 and 0.9 mg/ml, respectively. The stillborn male fetus was delivered at 22 weeks of gestation. Ultrasonography at 21 weeks had shown short limbs, micropenis, and possible hydrocephalus. Autopsy findings included fused eyelids, a nose with a single midline nasal pit and choanal atresia, high arched palate with broad alveolar ridges, postminimus of the left hand, postaxial polydactyly of the feet, syndactyly of the 2nd and 3rd, and 5th and 6th toes bilaterally, and nuchal edema. The external genitalia were female. The left lung was unilobate and the right lung was bilobate. The brain was a small solid mass (OFC 17 cm, more compatible with atelen/aprosencephaly than cebocephaly). The umbilical cord had two vessels. Case 3 was a term, live born infant. A short umbilical cord, cleft palate and abnormal oropharynx were noted at delivery. The infant had upward slanting palpebral fissures, small pupils, cataracts, a flattened nose, and a small tongue, mouth and mandible. The ears were angulated posteriorly. The infant had bilateral 2/3 toe syndactyly and polydactyly of both second toes, 3rd degree hypospadias, and unilateral cryptorchidism. The diagnosis was confirmed on the basis of hypocholesterolemia (19 mg/dl) and hyperdehydrocholesterolemia (180 mg/ ml). At 61 days of life, cholesterol treatment had increased the cholesterol levels to 55 mg/dl, but the infant had no psychomotor development. He developed hypertension, reflux, delayed gastric emptying, and suffered numerous upper and lower respiratory infections which ultimately lead to his death at age 7.5 months. Angle et al. [1998] reported an atypical case of RSH syndrome with hydrops, uncharacteristic facial appearance, and absence of 2/3 toe syndactyly in a 46,XY fetus who died at birth. Antenatal ultrasonography was abnormal and maternal uE3 levels were low. Cultured skin fibroblasts showed elevated 7DHC levels and abnormalities of cholesterol synthesis. In the atypical, but biochemically confirmed case of Seller et al. [1995] there was oligohydramnios with severe Pottersequence changes, webbing of axillae and crura (fetal akinesia), small right thumb and severe hypoplasia of right 5th finger, absence of left 5th finger, partial cutaneous syndactyly of toes 2/3 bilaterally, female external genitalia with testes and ducts, agenesis of right kidney and ureter and severe hypoplasia of the left, unilobate left lung, bilobed right lung, one pair of cervical ribs, and non-ossification or absence of vertebrae S4 and S5. Thus, postaxial oligodactyly may be the developmental equivalent of postaxial polydactyly. In the biochemically confirmed infant of Ness et al. [1997] there was, in addition to typical malformations, biventricular myocardial hypertrophy, PDA, aortic coarctation, bicuspid aortic valve, hepatomegaly with severe cholestasis, early septal fibrosis, marked bile staining of meninges with strikingly abnormal gyral pattern, mild hydrocephalus with porencephaly, absence of corpus callosum, and a hypoplastic cerebellum. Histologically there was an abnormality of cortical migration with four instead of six layers, and a severe lack of myelination. Also noted were depletion of thymocytes, enlarged hyperchromatic nuclei of the pancreatic islet cells *Correspondence to: Dr. Juliana G. Szakacs, Department of Pathology, University Utah School of Medicine, 50 N., Medical Drive, Salt Lake City, UT 84132. E-mail: Juliana.Szakacs@path.utah.edu