Prevalence of c.1559delT in ALPL, a common mutation resulting in the perinatal (lethal) form of hypophosphatasia in Japanese and effects of the mutation on heterozygous carriers

Hypophosphatasia (HPP) is an inherited disorder caused by mutations in ALPL that encodes an isozyme of alkaline phosphatase (ALP), TNSALP. One of the most frequent ALPL mutations is c.1559delT, which causes the most severe HPP, the perinatal (lethal) form (pl-HPP). c.1559delT has been found only in Japanese and its prevalence is suspected to be high; however, the allele frequency of c.1559delT in Japanese remains unknown. We designed a screening system for the mutation based on high-resolution melting curve analysis, and examined the frequency of c.1559delT. We found that the c.1559delT carrier frequency is 1/480 (95% confidence interval, 1/1562-1/284). This indicates that ～1 in 900?000 individuals to have pl-HPP caused by a homozygous c.1559delT mutation. In our analysis, the majority of c.1559delT carriers had normal values of HPP biochemical markers, such as serum ALP and urine phosphoethanolamine. Our results indicate that the only way to reliably detect whether individuals are pl-HPP carriers is to perform the ALPL mutation analysis.     

Utility of genetic testing for prenatal presentations of hypophosphatasia

Hypophosphatasia (HPP) is a rare inherited disease affecting bone and dental mineralization due to loss-of-function mutations in the ALPL gene encoding the tissue nonspecific alkaline phosphatase (TNSALP). Prenatal benign HPP (PB HPP) is a rare form of HPP characterized by in utero skeletal manifestations that progressively improve during pregnancy but often still leave symptoms after birth. Because the prenatal context limits the diagnostic tools, the main difficulty for clinicians is to distinguish PB HPP from perinatal lethal HPP, the most severe form of HPP.We previously attempted to improve genotype phenotype correlation with the help of a new classification of variants based on functional testing. Among 46 perinatal cases detected in utero or in the neonatal period for whose ALPL variants could be classified, imaging alone was thought to clearly diagnose severe lethal HPP in 35 cases, while in 11 cases, imaging abnormalities could not distinguish between perinatal lethal and BP HPP. We show here that our classification of ALPL variants may improve the ability to distinguish between perinatal lethal and PB HPP in utero.     

Perinatal hypophosphatasia: Radiology,pathology and molecular biology studies in a family harboring a splicing mutation (648+1A) and a novel missense mutation (N400S) in the tissue‐nonspecific alkaline phosphatase (TNSALP) gene

We report on a postmortem diagnosis of perinatal lethal hypophosphatasia, an inborn error of metabolism characterized by a liver/bone/kidney alkaline phosphatase (ALP)‐related defective bone mineralization due to mutations in the tissue‐nonspecific alkaline phosphatase (TNSALP) gene. Radiological and pathological studies identified a perinatal lethal hypophosphatasia showing a generalized bone mineralization defect including asymmetry of the cervical vertebral arches in a 22 +4 weeks' gestation fetus. Both parents revealed low serum ALP activities supporting the diagnosis. Sequencing analysis of the TNSALP gene showed two heterozygous mutations, 648+1A, a mutation affecting the donor splice site in exon 6, and N400S, a novel missense mutation in exon 11, located near the active site and very close to histidins 364 and 437, two crucial residues of the active site. Sequencing of exons 6 and 11 in the parents showed that 648+1A was from maternal origin and N400S from paternal origin. DNA‐based prenatal testing in the subsequent pregnancy following a chorionic villous sampling performed at 10 weeks of gestation showed no mutation and a healthy infant was born at term. © 2001 Wiley‐Liss, Inc.     

Monoamniotic twins with one fetal anencephaly and cord entanglement diagnosed with three dimensional ultrasound at 14 weeks of gestation

A 29-year-old pregnant woman with parity 0-0-0-0 was diagnosed with monoamniotic twin pregnancy discordant for anencephaly at 14 weeks gestation. Umbilical cord entanglement, which is an important cause of fetal death in monoamniotic twins, was confirmed by three-dimensional ultrasound. Cesarean section was performed at 34 weeks of gestation, and the normal newborn infant was discharged without any complications. We report a case of monoamniotic twin pregnancy discordant for anencephaly and diagnosed with cord entanglement by three-dimensional ultrasound at 14 weeks of gestation, and now report it along with a literature review.     

Heritability of Obesity‐related Phenotypes and Association with Adiponectin Gene Polymorphisms in the Chinese National Twin Registry

The purpose of this study was to estimate the heritability of obesity‐related phenotypes and investigate the association of adiponectin gene polymorphisms +45T>G and +276G>T with these measures in Chinese twins. 1260 twin pairs were recruited from two cities through the Chinese National Twin Registry System from 2001 to 2005. Two SNPs at the adiponectin locus (+45T>G and +276G>T) were genotyped. Structural equation modeling (SEM) was used to estimate heritability and the best‐fitting variance component model. The regular association among all twins was analysed with generalised estimating equations (GEE). Sib‐transmission/disequilibrium test (TDT) within dizygotic (DZ) twin pairs discordant for their genotype was performed using SEM. Additive genetic, common and unique environmental (ACE) model‐based heritability of body mass index (BMI) was 61%, while additive genetic and unique environmental (AE)‐model‐based heritability of waist circumference (WC) and waist‐hip ratio (WHR) were 75% and 61%, respectively. There was no association of adiponectin gene +45T>G and +276G>T genotypes with obesity‐related phenotypes in all twins or discordant DZ twins. Our twins data did not support that there was an association between adiponectin gene polymorphisms +45T>G and +276G>T and the obesity‐related phenotypes. Further studies are required to better understand the role of adiponectin gene polymorphisms in obesity.     

An Emerging Female Phenotype with Loss‐of‐Function Mutations in the Aristaless‐Related Homeodomain Transcription Factor ARX

The devastating clinical presentation of X‐linked lissencephaly with abnormal genitalia (XLAG) is invariably caused by loss‐of‐function mutations in the Aristaless‐related homeobox (ARX) gene. Mutations in this X‐chromosome gene contribute to intellectual disability (ID) with co‐morbidities including seizures and movement disorders such as dystonia in affected males. The detection of affected females with mutations in ARX is increasing. We present a family with multiple affected individuals, including two females. Two male siblings presenting with XLAG were deceased prior to full‐term gestation or within the first few weeks of life. Of the two female siblings, one presented with behavioral disturbances, mild ID, a seizure disorder, and complete agenesis of the corpus callosum (ACC), similar to the mother's phenotype. A novel insertion mutation in Exon 2 of ARX was identified, c.982delCinsTTT predicted to cause a frameshift at p.(Q328Ffs^*37). Our finding is consistent with loss‐of‐function mutations in ARX causing XLAG in hemizygous males and extends the findings of ID and seizures in heterozygous females. We review the reported phenotypes of females with mutations in ARX and highlight the importance of screening ARX in male and female patients with ID, seizures, and in particular with complete ACC.     

Mutations in CRLF1 cause familial achalasia

We here report a family from Libya with three siblings suffering from early onset achalasia born to healthy parents. We analyzed roughly 5000 disease‐associated genes by a next‐generation sequencing (NGS) approach. In the analyzed sibling we identified two heterozygous variants in CRLF1 (cytokine receptor‐like factor 1). Mutations in CRLF1 have been associated with autosomal recessive Crisponi or cold‐induced sweating syndrome type 1 (CS/CISS1), which among other symptoms also manifests with early onset feeding difficulties. Segregation analysis revealed compound heterozygosity for all affected siblings, while the unaffected mother carried the c.713dupC (p.Pro239Alafs*91) and the unaffected father carried the c.178T>A (p.Cys60Ser) variant. The c.713dupC variant has already been reported in affected CS/CISS1 patients, the pathogenicity of the c.178T>A variant was unclear. As reported previously for pathogenic CRLF1 variants, cytokine receptor‐like factor 1 protein secretion from cells transfected with the c.178T>A variant was severely impaired. From these results we conclude that one should consider a CRLF1‐related disorder in early onset achalasia even if other CS/CISS1 related symptoms are missing.     

A case of lethal hypophosphatasia providing new insights into the perinatal benign form of hypophosphatasia and expression of the ALPL gene

Hypophosphatasia is a rare inherited bone disease caused by mutations in the alkaline phosphatase liver-type gene (ALPL) gene, with extensive allelic heterogeneity leading to a range of clinical phenotypes. We report here a patient who died from severe lethal hypophosphatasia, who was compound heterozygous for the mutation c.1133A>T (D361V) and the newly detected missense mutation c791A>G, and whose parents were both healthy. Because the c.1133A>T (D361V) mutation was previously reported to have a dominant-negative effect and to be responsible for the uncommon perinatal benign form of the disease, we studied the expression of the ALPL gene in this family. Analysis at the messenger RNA (mRNA) level, both quantitative and qualitative, showed that the paternal c.1133A>T (D361V) mutation was associated with over-expression of the ALPL gene and that the maternal c.791A>G mutation lead to complete skipping of exon 7. The results provide an explanation of the lethal phenotype in the patient where the two ALPL alleles are non-functional and in the asymptomatic father where over-expression of the normal allele could counteract the effect of the c.1133A>T (D361V) mutation by providing an increased level of normal mRNA. This may also explain the variable expression of hypophosphatasia observed in parents of patients with the perinatal benign form.     

低磷酸酶血症一家系组织非特异性碱性磷酸酶TNSALP基因突变分析

 目的 对1例儿童型低磷酸酶血症（HPP）患者及家系进行临床分析及基因突变检测,以探讨HPP的致病机制。方法 针对1例罕见的HPP患者的典型临床特点,进行实验室检验及影像学检查。进而收集患者及其亲属外周血,提取基因组DNA。针对ALPL基因12个外显子及附近内含子区合成引物,经PCR扩增后,直接对产物测序检测突变。结果 显示患者血碱性磷酸酶水平显著降低,骨骼具有佝偻病样改变;患者ALPL基因存在c.18delA及c.G407C两种突变。前者所致移码突变使得翻译提前终止,形成的截短蛋白 (p.V7Yfs18X)丧失了发挥酶活性及骨骼矿化作用的重要区域;而c.G407C导致其编码的氨基酸由精氨酸变为脯氨酸（R136P）。进一步检索PubMed及ALPL基因突变数据库,以上突变在国内外均未见报道。临床表现正常的患者母亲及祖母、父亲分别携带c.18delA和c.G407C突变,该家系符合常染色体隐性遗传。结论 ALPL基因c.18delA和c.G407C两种新突变,与HPP临床表现密切相关。     

Desbuquois dysplasia type I and fetal hydrops due to novel mutations in the CANT1 gene

We report on three hydropic fetuses of 17, 22 and 25 gestational weeks from three distinct families presenting with Desbuquois dysplasia type 1. All fetuses showed brachymelia and characteristic dysmorphic features. X-ray studies revealed δ-shaped extraphalangeal bones and disease-specific prominence of the lesser trochanter, varying in severity with fetal age. Early lethal manifestation of the disorder was reflected in lung hypoplasia and in early death of similarly affected siblings in cases 1 and 2. All families were German Caucasians by descent. Sequence analysis of the CANT1 gene revealed two frameshift mutations, c.228_229insC and c.277_278delCT, in homozygous and compound heterozygous configuration, respectively, and a homozygously novel missense mutation, c.336C>A (p.D112E), located within a highly conserved region of exon 2. Haplotype analyses by high-resolution single-nucleotide polymorphism array showed that the haplotype associated with c.228_229insC may be traced to a single founder in the German population.     

Perinatal hypophosphatasia: radiology, pathology and molecular biology studies in a family harboring a splicing mutation (648+1A) and a novel missense mutation (N400S) in the tissue-nonspecific alkaline phosphatase (TNSALP) gene.

We report on a postmortem diagnosis of perinatal lethal hypophosphatasia, an inborn error of metabolism characterized by a liver/bone/kidney alkaline phosphatase (ALP)-related defective bone mineralization due to mutations in the tissue-nonspecific alkaline phosphatase (TNSALP) gene. Radiological and pathological studies identified a perinatal lethal hypophosphatasia showing a generalized bone mineralization defect including asymmetry of the cervical vertebral arches in a 22 +4 weeks' gestation fetus. Both parents revealed low serum ALP activities supporting the diagnosis. Sequencing analysis of the TNSALP gene showed two heterozygous mutations, 648+1A, a mutation affecting the donor splice site in exon 6, and N400S, a novel missense mutation in exon 11, located near the active site and very close to histidins 364 and 437, two crucial residues of the active site. Sequencing of exons 6 and 11 in the parents showed that 648+1A was from maternal origin and N400S from paternal origin. DNA-based prenatal testing in the subsequent pregnancy following a chorionic villous sampling performed at 10 weeks of gestation showed no mutation and a healthy infant was born at term.     

A homozygous variant in growth and differentiation factor 2 (GDF2) may cause lymphatic dysplasia with hydrothorax and nonimmune hydrops fetalis

The etiology of nonimmune hydrops fetalis is extensive and includes genetic disorders. We describe a term‐born female neonate with late onset extensive nonimmune hydrops, that is, polyhydramnios, edema, and congenital bilateral chylothorax. This newborn was successfully treated with repetitive thoracocentesis, total parenteral feeding, octreotide intravenously and finally surgical pleurodesis and corticosteroids. A genetic cause seemed plausible as the maternal history revealed a fatal nonimmune hydrops fetalis. A homozygous truncating variant in GDF2 (c.451C>T, p.(Arg151*)) was detected with exome sequencing. Genetic analysis of tissue obtained from the deceased fetal sibling revealed the same homozygous variant. The parents and two healthy siblings were heterozygous for the GDF2 variant. Skin and lung biopsies in the index patient, as well as the revised lung biopsy of the deceased fetal sibling, showed lymphatic dysplasia and lymphangiectasia. To the best of our knowledge, this is the first report of an association between a homozygous variant in GDF2 with lymphatic dysplasia, hydrothorax and nonimmune hydrops fetalis.     

Ontogenetic allometry of ossified fetal limb bones.

The skeletal components of fetal limbs change in both size and shape throughout gestation. Relative growth of different bones, as well as differences between homologous bones of the upper and lower limbs, are not well known for all stages of fetal development. This study used human fetal skeletal material (N = 57) ranging in age from 19 to 40 weeks gestation, and in body mass from 290g to 4650g. Measurements of maximal length and minimal width were taken from the six major long bones: femur, tibia, fibula, humerus, radius, and ulna. These data were log transformed, and growth rates determined from least squares regression of bone length or bone width on body mass and on crown-to-rump length. The results indicated that growth rates are equivalent among bones within a limb, whereas homologous bones in the upper and lower limb grew at different rates. In general, the upper limb bones display negative allometry and the lower limb bones display isometric growth in relation to body mass and crown-to-rump length. Further, there was no difference between growth rates of length and width relative to body mass. The negative slopes of upper-to-lower limb bones in relation to mass confirm the conclusion that lower limb bones grow faster than the upper limb bones from 19 weeks gestation to birth. These results, together with results from similar studies of other periods of fetal development, provide a unified picture of the prenatal growth in human skeletal limbs.     

Clinical variability and novel mutations in the NHEJ1 gene in patients with a Nijmegen breakage syndrome‐like phenotype

We have previously shown that mutations in the genes encoding DNA Ligase IV (LIGIV) and RAD50, involved in DNA repair by nonhomologous‐end joining (NHEJ) and homologous recombination, respectively, lead to clinical and cellular features similar to those of Nijmegen Breakage Syndrome (NBS). Very recently, a new member of the NHEJ repair pathway, NHEJ1, was discovered, and mutations in patients with features resembling NBS were described. Here we report on five patients from four families of different ethnic origin with the NBS‐like phenotype. Sequence analysis of the NHEJ1 gene in a patient of Spanish and in a patient of Turkish origin identified homozygous, previously reported mutations, c.168C>G (p.Arg57Gly) and c.532C>T (p.Arg178Ter), respectively. Two novel, paternally inherited truncating mutations, c.495dupA (p.Asp166ArgfsTer20) and c.526C>T (p.Arg176Ter) and two novel, maternal genomic deletions of 1.9 and 6.9 kb of the NHEJ1 gene, were found in a compound heterozygous state in two siblings of German origin and in one Malaysian patient, respectively. Our findings confirm that patients with NBS‐like phenotypes may have mutations in the NHEJ1 gene including multiexon deletions, and show that considerable clinical variability could be observed even within the same family. Hum Mutat 31:1059–1068, 2010. © 2010 Wiley‐Liss, Inc.     

Clinical and molecular analysis in Papillon–Lefèvre syndrome

Papillon–Lefèvre syndrome (PLS; MIM#245000) is a rare recessive autosomal disorder characterized by palmar and plantar hyperkeratosis, and aggressively progressing periodontitis leading to premature loss of deciduous and permanent teeth. PLS is caused by loss‐of‐function mutations in the CTSC gene, which encodes cathepsin C. PLS clinical expressivity is highly variable and no consistent genotype–phenotype correlation has been demonstrated yet. Here we report the clinical and genetic features of five PLS patients presenting a severe periodontal breakdown in primary and permanent dentition, hyperkeratosis over palms and soles, and recurrent sinusitis and/or tonsillitis. Mutation analysis revealed two novel homozygous recessive mutations (c.947T>C and c.1010G>C) and one previous described homozygous recessive mutation (c.901G>A), with parents carrying them in heterozygous, in three families (four patients). The fourth family presented with the CTSC c.628C>T mutation in heterozygous, which was inherited maternally. Patient carrying the CTSC c.628C>T mutation featured classical PLS phenotype, but no PLS clinical characteristics were found in his carrier mother. All mutations were found to affect directly (c.901G>A, c.947T>C, and c.1010G>C) or indirectly (c.628C>T, which induces a premature termination) the heavy chain of the cathepsin C, the region responsible for activation of the lysosomal protease. Together, these findings indicate that both homozygous and heterozygous mutations in the cathepsin C heavy chain domain may lead to classical PLS phenotype, suggesting roles for epistasis or gene–environment interactions on determination of PLS phenotypes.     

Ultrastructural Abnormalities in Bone and Calcifying Cartilage in Two Siblings with a Newly Described Recessive Lethal Chondrodysplasia

Ultrastructural abnormalities in bone and calcifying cartilage are presented for a recently identified lethal chondrodysplasia. Two siblings, aged 20 and 30 weeks of gestation, showed severe short-limb dwarfism and histologically distinct, highly disorganized masses of cartilage, bone, and mesenchymal tissue in the long bones. Regions of inappropriate cartilage calcification showed unusual, electron-dense, amorphous islands of mineralization and larger, less dense, layered calcified masses that occasionally entrapped chondrocytes. Bone abnormalities included abnormal cartilage-bone transition at the growth plate, general bone matrix disorganization due to irregularly oriented bundles of collagen, mineral crystals on the osteocyte lacunar rim, and accumulations of thickened collagen fibrils, also along the osteocyte lacunar rim. These findings point to abnormal calcification and mineralization distinct from those seen in other reported skeletal dysplasias. These abnormalities are associated with an anarchic distribution of mesen-chymelike tissue infiltrating the cartilage and bone.     

FETAL ASCITES A Report of 3 Autopsy Cases

Three rare autopsy cases of fetal ascites were presented and the etiology of each case was described. Case 1 was a male neonate, delivered by cesarean section at 32 weeks’gestation, and died of respiratory failure. The abdomen was remarkably distended with 1020 ml of ascites. The etiology of Case 1 remained unknown even after macroscopic and microscopic examinations. We considered this as “idiopathic” fetal ascites. Case 2 was a female neonate, delivered at 31 weeks’gestation, with marked abdominal distension and cyanosis. Autopsy revealed 435 ml of ascites, and she was considered to have had “polysplenia syndrome” with cardiovascular malformations. Intrauterine heart failure due to cardiac anomalies was thought to be the cause of this ascites. In case 3 embryotomy was carried out under the diagnosis of fetal ascites by ultrasound examination at 22 weeks’gestation. An urachal cyst connected to the dilated urinary bladder and deficiency of musculature of the abdominal wall composed of loose connective tissue with calcification were observed. The abdominal wall was ruptured and 1,960 ml of ascites was measured. Polycystic kidney with renal dysplasia was also found. Case 3 showed “Prune-Berry syndrome” and fetal ascites may have arisen from these anomalies.     

水肿胎儿血液生化指标分析

目的检测水肿胎儿（包括Bart′s、染色体异常、病毒感染、同种免疫性及其他原因所致水肿胎儿）血液生化指标并与相同孕周胎儿血液生化指标比较,分析各种原因所致水肿胎儿血液生化指标变化,探讨水肿胎儿病因与病理,为胎儿疾病诊断与治疗提供线索和依据。方法应用Bayer1650全自动生化分析仪对66例孕19～35周水肿单活胎儿脐静脉穿刺所取脐血进行：碱性磷酸酶（ALP）、谷草转氨酶（AST）、谷丙转氨酶（ALT）、总胆红素（TB）、直接胆红素（DB）、间接胆红素（IB）、总蛋白（TP）、白蛋白（ALB）、球蛋白（GLB）、r-谷氨酰基转酞酶（r-GT）、羟丁氨酸脱氢酶（HBDH）、乳酸脱轻酶（LDH）、肌酸磷酸激酶（CK）、肌酸磷酸激酶同工酶-1（CK-MB）等项生化指标检测,同时进行染色体,TORCH,胎儿血红蛋白和血常规等检查;并按染色体、TORCH、Bart′s、不明原因的水肿胎儿分为四类和按孕周分组统计与同孕周正常胎儿比较。结果除外Bart′s水肿胎其余原因（包括染色体、TORCH、和其他原因）所致水肿胎的心、肝酶及蛋白等水平均与与同孕周正常胎儿的水平有显著差异。结论胎儿在水肿的病理状态时存在血液生化指标异常,可能是水肿的原因或是后果,可作为水肿胎儿诊断与评估及治疗效果观察的参考指标。     

106例胎儿血生化指标分析研究

目的了解胎儿血液生化指标特点及在各孕周变化规律,为胎儿宫内代谢异常的诊断与治疗提供参考依据.方法应用日立7170全自动生化分析仪对106例孕19～38w胎儿脐血进行血液尿素氮(BUN)、二氧化碳结合力(CO2)、肌酸磷酸激酶同工酶-1(CK-MB)等18项生化指标检测,胎儿脐血取自B超引导下母腹脐静脉穿刺产前诊断其结果正常胎儿,并经胎血鉴定,各指标按孕周分组计算均值,经统计学分析.结果发现胎儿各孕周组血液生化指标中BUN、CO2、Cr、UA、ALP、AST、ALT、TB、HBDH、LDH、CK、CK-MB等代表心、肾功能指标均无显著差异(P＞0.05),代表肝功能的DB、IB、TP、ALB、GLB、r-GT均值在胎儿各孕期随孕周增加升高,有统计学意义(P＜0.05).结论胎儿生化指标有独特的参考范围心、肾功能指标在整个孕期变化不大,肝功能各项指标随孕周曾加.     

Exome sequencing reveals three homozygous missense variants in SNRPA in two sisters with syndromic intellectual disability

Splicing‐related gene mutations might affect the expression of a single gene or multiple genes and cause clinically heterogeneous diseases. With the advent of next‐generation sequencing, several splicing gene mutations have been exposed, yet most major spliceosome genes have no reports of germline mutations and therefore, their effects are largely unknown. We describe the previously unreported concurrence of intellectual disability, short stature, poor speech, and minor craniofacial and hand anomalies in 2 female siblings with 3 homozygous missense variants in SNRPA (a component of the U1 small nuclear ribonucleoprotein complex) characterized by homozygosity mapping and whole exome sequencing. Combined, c.97A>G, c.98T>C, and c.100T>A, in exon 2 of SNRPA lead to p.Ile33Ala and p.Phe34Ile exchanges, which were predicted in silico to be deleterious. Although both patients exhibited some clinical features seen in other spliceosomal disorders, their complete clinical phenotype appears to be rather uncommon, a finding that may further support the notion that mutations in components of the major spliceosome do not strictly lead to the same syndromes/phenotypes.