Identification of a Gene for Renal-Hepatic-Pancreatic Dysplasia by Microarray-Based Homozygosity Mapping

Abstract

We have investigated a family where two siblings had a developmental disorder associated with polycystic dysplastic kidney disease that was incompatible with postnatal survival. Additional features observed were ductal plate malformation in the liver, dysplasia of the pancreas, and (in one individual) complete situs inversus and polymicrogyria of the cingulate gyri. The autopsy findings were compatible with renal-hepatic-pancreatic dysplasia, a condition with unknown genetic cause at the time of autopsy but with similarities to the Meckel-Gruber/Joubert group of recessive ciliopathies. Consanguinity between the parents made it likely that the mutated gene (with known or potential function in cilia) was located within a rather large region of homozygosity in the affected individuals (identical by descent). Using genetic markers (50K single nucleotide polymorphism microarrays), we found a single large homozygous region of 21.16 Mb containing ∼200 genes on the long arm of chromosome 3. This region contained two known ciliopathy genes: NPHP3 (adolescent nephronophthisis) and IQCB1 (NPHP5), which is associated with Senior-Löken syndrome. In NPHP3, homozygosity for a deletion of the conserved splice acceptor dinucleotide (AG) preceding exon 20 was found. Our finding confirms the recent report that NPHP3-null mutations cause renal-hepatic-pancreatic dysplasia. Also, our case illustrates that genes for rare and genetically heterogeneous recessive conditions may be identified by homozygosity mapping using single nucleotide polymorphism arrays in the routine clinical setting.

The combination of cystic kidneys and hepatic fibrosis in a fetus or newborn is usually indicative of infantile autosomal recessive polycystic kidney disease. This condition is often lethal because of oligohydramnios with subsequent pulmonal hypoplasia. In addition to autosomal recessive polycystic kidney disease, cystic kidneys and hepatic periportal fibrosis with or without bile-duct anomalies (ductal plate malformation) may also be found in other neonatal lethal syndromes that belong to the rapidly increasing group of inherited diseases called ciliopathies.¹ This group includes Meckel-Gruber syndrome, severe forms of autosomal dominant polycystic kidney disease, Joubert syndrome, and Bardet-Biedl syndrome (Table 1). Because of the widespread importance of primary cilia for normal organ development, ciliopathies may have quite variable phenotypes and degree of organ involvement.¹ Since cilia are also involved in left-right body asymmetry determination, ciliopathies are also a cause of lateralization defects, including situs inversus. Because the recurrence risk for a couple with a child with a lethal ciliopathy is 25% in most cases, a DNA-based prenatal diagnostic test that can be applied at an early time point in subsequent pregnancies is often desired. However, genetic heterogeneity and variable expression of different mutations in the same gene complicate molecular diagnostics (Table 1). Thus, mutations in CEP290 may cause isolated retinal disease (Leber's congenital amaurosis), Joubert syndrome, or Meckel-Gruber syndrome,² and RPGRIP1L mutations are seen in both Joubert and Meckel-Gruber syndromes.³ Antenatal lethal cases with polycystic kidneys and ductal plate malformation but without encephalocele may be caused by mutations in the Bardet-Biedl genes BBS2, BBS4, or BBS6⁴ or by mutations in the Meckel-Gruber genes MKS1, MKS3, and CEP290⁵ (Table 1).

Table 1.

Differential Diagnoses in Lethal Cystic Kidney Disease

	RHPD	Autosomal recessive polycystic kidney disease	Autosomal dominant polycystic kidney disease*	Meckel-Gruber	Joubert	Bardet-Biedl	Multicystic dysplastic kidneys,† bilateral
Polycystic kidneys	Cystic dysplasia	Small, uniform corticomedullary cysts	Variable picture, whole nephron affected	Cystic dysplasia	Cystic dysplasia, Nephronophthisis	Cystic dysplasia or medullary cysts	Cystic dysplasia
Ductal plate malformation	+	+	(+)	+	+	+
Pancreatic dysplasia	++			(+)
Situs inversus/ heterotaxia	+			+	+	+
Encephalocele, Neural tube defects				++	(+)
Polydactyly	(+)			+	+	+
Molar tooth sign				(+)	++
Genes mutated	NPHP3	PKHD1	PKD1, PKD2	MKS1 TMEM67 RPGRIP1L CEP290 CC2D2A	AHI1 NPHP1 ARL13B TMEM67 RPGRIP1L CEP290 CC2D2A	BBS2 BBS4 BBS6 MKS1 TMEM67 CEP290
Inheritance	Autosomal recessive	Autosomal recessive	Autosomal dominant	Autosomal recessive	Autosomal recessive	Autosomal recessive	Sporadic

Typical findings in lethal forms of these conditions are listed but not symptoms known to occur in surviving individuals, like for example, retinitis pigmentosa in Bardet-Biedl or Joubert syndrome.

^*Rarely, autosomal dominant polycystic kidney disease may present antenatally or perinatally with severe cystic kidney disease and ductal plate malformation.

^†Multicystic dysplastic kidneys: Sporadic condition, low risk of recurrence, although an autosomal dominant condition with variable penetrance have been described previously.

Renal-hepatic-pancreatic dysplasia (RHPD) is another lethal condition reminiscent of a ciliopathy, first described by Ivemark et al⁶ and sometimes referred to as Ivemark II syndrome (OMIM number 208540). In addition to the signs mentioned above, cystic dysplasia of the pancreas and situs inversus or other lateralization defects can be seen. Here, we describe two siblings with severe malformations compatible with RHPD, born to remotely consanguineous parents. At the time of diagnosis, the molecular genetic cause of RHPD was unknown. Because a search for a causative gene among the known ciliopathy genes is expensive, time-consuming and possibly inconclusive, we used microarrays with 50,000 single nucleotide polymorphisms (Affymetrix 50K) to identify a chromosomal locus for such a gene, with the hypothesis that the affected siblings had inherited the same mutation from both the father and the mother (ie, the mutation was identical by descent from the same ancestor).⁷ This method is called homozygosity mapping.⁷ The affected siblings will also be homozygous for all of the genetic markers in close vicinity of the mutation, and the size of the homozygosity region is dependent on how closely related the parents are.

Materials and Methods

Subjects

DNA was obtained from the affected deceased male and female fetus, three healthy siblings (one girl and two boys), both parents and three grandparents. The parents were double fourth cousins (Figure 1, only one inbreeding loop is shown). The adults gave informed consent to the investigation and publication of the results.

Figure 1.

Pedigree and haplotypes in the candidate region on chromosome 3q. Selected single nucleotide polymorphisms and their distance in megabases from the end of the short arm of chromosome 3 are shown in the left columns in consecutive order. Parental haplotypes have been given different colors, with the shared (ancestral) haplotype in red. The boundaries of the candidate region are indicated by dashed lines. The NPHP3 gene is located 133.88 to 133.92 Mb and the IQCB1 gene 122.97 to 123.03 Mb from the start of chromosome 3 (3pter).

Pathological Examination

A complete perinatal autopsy was performed in both cases. In case 2, the small 16-weeks fetus was examined in part under a dissection microscope (Olympus SZ40; Olympus Medical Systems Corp., Tokyo, Japan). Formalin-fixed tissue specimens from fetal viscera and placenta were processed in paraffin blocks and cut for microscopy according to routine laboratory procedures.

Genotyping and Homozygosity Mapping

The expected size and number of regions of homozygosity in the child may be estimated when the relationship between the parents is known. In this case, we expected only one candidate region of ∼16 cM.⁸ Whole-genome scan was performed using the Affymetrix 50K Chip (Affymetrix, Santa Clara, CA), and the data were exported and treated for further analysis by use of the programs GTYPE (Afymetrix) and Progeny Lab (Progeny Software, South Norfolk, UK). Regions of homozygosity were identified using the PLINK software.⁹ Physical marker positions are given according to NCBI Build 36.3 (http://www.ncbi.nlm.nih.gov/mapview, accessed March 1, 2008).

DNA Sequencing and Mutation Detection

PCR primers for amplification of exons and flanking intronic sequences of both NPHP3 and IQCB1 were designed using the Oligo 6.3 program (National Bioscience, Plymouth, MN). Reference sequences for NPHP3 and IQCB1 are NM_153240 and NM_014642 respectively. PCR products were treated with Shrimp alkaline phosphatase/exonuclease I (Amersham Biosciences, Piscataway, NJ) and sequenced using the PRISM BigDye Terminator kit (version 1.1) and an ABI 3730 Genetic Analyzer (Applied Biosystems, Foster City, CA). Data analysis was assisted by use of the Seqscape software (Applied Biosystems).

RNA Analyses

Blood from the parents and normal controls were collected in Tempus tubes (Applied Biosystems). Total RNA was purified using the ABI 6100 system (Applied Biosystems), and the quality of the RNA was analyzed using the Experion system (Bio-Rad, Hercules, CA). cDNA synthesis was performed using the TaqMan Reverse Transcription kit (Applied Biosystems). PCR amplification of the cDNA (exons 19 to 21) was performed using the 5′-CAGATGAACTCCCGTGGC-3′ and 5′-AAAGGGCAAATCCGTACAAGT-3′ primers. The PCR products were cloned into the pCR2.1 vector using the Invitrogen TOPO TA Cloning Kit (Invitrogen, Carlsbad, CA). Clones were sequenced using the Prism BigDye terminator version 1.1 kit and the 3730 Genetic Analyzer (Applied Biosystems).

Immunohistochemistry

Immunohistochemistry was performed on formalin-fixed and paraffin-embedded kidney tissue sections. Antigen retrieval was performed for 15 minutes in citraconic anhydride in a pressure cooker. Sections were incubated with nephrocystin-3 antibody (N-18, 1/500; Santa Cruz Biotechnology, Santa Cruz, CA). Bound antibodies were detected by a streptavidin-biotin kit (LSAB; DakoCytomation, Carpinteria, CA) or EnVision detection kit (DakoCytomation) in combination with a link antibody from rabbit. Diaminobenzidine was used for visualization of immunoreactivity, followed by hematoxylin for nuclear counterstaining. In the negative controls, the primary antibody was omitted. Age-matched normal kidney tissue served as positive control.

Results

Clinical Findings

Case 1: Boy Delivered by Caesarian Section in Week 34

This was the couple's first pregnancy. Caesarean section was performed in the 34th gestational week because of intrauterine growth retardation and oligohydramnios. Birth weight was 1775 g. The baby boy died 1 day after delivery due to respiratory insufficiency. An autopsy revealed typical Potter facies with hypertelorism, a beak-like nose, low-set ears, a short neck, and a short sternum. Lungs and kidneys were hypoplastic. The lungs weighed 13.9 g (left) and 16.0 g (right) (reference weight is 19.8 and 22.6 g, respectively). The kidneys weighed 3.7 g (left) and 3.6 g (right) (reference weight is 9.5 and 9.1 g, respectively¹⁰). The kidneys showed complete loss of normal architecture with multiple small cysts. The pancreas seemed enlarged and fibrotic (pancreas weight, 4.6 g; reference weight, 4.0 g). Microscopic examination of the kidneys showed primitive tubules and tubular cysts in a mesenchymal stroma with rare malformed glomeruli and foci of cartilage consistent with renal dysplasia (Figure 2, A and B). The ureter was thinner than normal, but a tiny lumen was revealed by microscopic investigation. There was portal fibrosis in the liver, paucity of bile ducts, and persistence of embryonic bile duct structures in the ductal plate (Figure 2C). The pancreas showed slightly disordered lobular architecture, irregular ducts, focal atrophy of acini, pronounced fibrosis, and scattered islets of Langerhans (Figure 2D). Investigation of the brain showed normal results.

Figure 2.

Case 1. Kidneys with cystic dysplasia (A and B). Primitive tubules surrounded by mesenchymal tissue with foci of cartilage and a malformed glomerulus (B). Liver with portal fibrosis and paucity of bile ducts; this portal area contains vessels, but no ducts (C). The pancreatic tissue shows marked fibrosis and dysplastic ducts. Two islets of Langerhans are also seen (D). A and B: H&E; C and D: trichrome. Scale bars: 5 mm (A) and 200 μm (B–D).

Case 2: Elective Abortion of Female Fetus at 16 Weeks of Gestation

This was the couple's sixth pregnancy. The female fetus weighed 142 g, crown-heel length was 18.5 cm, crown-rump length was 12.5 cm, foot length was 2.2 cm, and head circumference was 12.5 cm. There were dysmorphic features consistent with Potter facies and bilateral symmetrical flexion contractures of multiple joints (elbows, wrists, hips, and knees; Figure 3A). Organ dissection revealed situs inversus of thoracic and abdominal viscerae. The heart was dextroposed (Figure 3A) with the left atrium, ventricle, and ascending aorta on the right side, and with the right atrium, ventricle, and pulmonary truncus on the left side. The right lung had two lobes and the left three lobes. Apart from this mirror imaging of the heart and lungs, no malformations were found in the thoracic organs or vessels. In the abdomen (Figure 3B), the liver and gall bladder were located to the left, and the pancreas and spleen to the right, again consistent with situs inversus. The liver weighed 7.4 g (reference weight, 5.8 g¹⁰). The ileocoecal valve with the appendix and ascending colon was identified at the left side and the descending colon at the right side of the abdomen. The kidneys were symmetric and enlarged, with a combined weight of 4.5 g (reference weight, 0.7 g¹⁰) and had a spongy cut surface. Macroscopic and microscopic examination revealed the presence of a renal pelvis, and the ureteres were well formed, ending in a small urinary bladder. The adrenal glands had normal position and size. In the pelvis, a small uterus and bilateral ovaries were identified. The external surface of the brain was normal. There were no signs of hydrocephalus, but the lateral ventricles had an abnormal triangular configuration. Weight and appearance of the placenta were unremarkable.

Figure 3.

Case 2. Situs inversus with dextroposition of the heart (A, asterisk) and sinistroposed liver with gall bladder (B, white arrow). Flexion contractures of the elbows and wrists (A). Curvatura major of the stomach to the right (B, arrowhead) and ileocoecal segment with appendix on the left side of the abdomen (B, red arrow). Hepatic dysplasia with abnormally branching medium-sized bile ducts (Masson trichrome) (C). Brain: The cingulate gyri demonstrated polymicrogyria (arrow) (H&E) (D). Scale bars: 100 μm (C) and 2000 μm (D).

On microscopy, the kidneys showed features consistent with renal dysplasia, containing abundant epithelium-lined cysts and tubular structures in a mesenchymal soft tissue with a few abortive glomeruli (similar to case 1; Figure 2, A and B). In the liver, the lobular tissue was unremarkable with extramedullary hematopoiesis. There were, however, features consistent with ductal plate malformation with expanded portal tracts with paucity of small bile ducts and complex abnormally branched medium-sized bile ducts (Figure 3C). The pancreas showed dysplastic features with slightly dilated ductal structures with scarce lobular differentiation in an abundant mesenchymal stroma. In larger ducts, there seemed to be exaggerated epithelial proliferation, in part with papillary fronds filling up the ductal lumina. Rounded-cell clusters consistent with immature islets were identified. Scattered lymphoid infiltrates were seen. In the brain, the cingulate gyri demonstrated excessively folded cortical miniature ribbons with thin and partly fused convolutions, consistent with polymicrogyria (Figure 3D).

Molecular Genetic Analysis

The only region of homozygosity with a size >1 Mb in the affected siblings was located in the middle of the long arm of chromosome 3 in domains 3q13.2-q22.2. The candidate region was 21.16 Mb, limited by the markers rs3773684 (114.82 Mb from 3pter) and rs2138212 (135.98 Mb from 3pter) (Figure 1). This region contained ∼200 genes, including two genes known to cause ciliopathies: NPHP3, linked to adolescent nephronophthisis, and IQCB1 (NPHP5), associated with Senior-Löken syndrome. Both genes were sequenced, and in NPHP3, the affected boy was homozygous for a deletion of the splice acceptor (AG) preceding exon 20, c.2694-2_2694-1delAG (Figure 4) (nomenclature: http://www.hgvs.org/rec.html, accessed March, 1, 2008). RNA analysis showed that this caused activation of a cryptic splice signal in intron 19 that led to out-of-frame inclusion of 19 bp of intronic sequence, followed by a premature stop codon (Figure 5). An immunohistochemical stain for nephrocystin-3 in tissue from cases 1 (Figure 6) and 2 showed no or very weak staining in the primitive tubules of the dysplastic kidneys, whereas age-matched normal kidney tissue showed distinct granular staining in collecting ducts (16 weeks; data not shown) and in proximal tubules in the inner cortex (34 weeks; Figure 6).

Figure 4.

The mutation c.2694-2_2694-1delAG. The normal sequence is shown on top (wild type (WT)), with the fetal sequence below. The fetus was homozygous for the mutation.

Figure 5.

Effect of the c.2694-3_2694-1delAG mutation on the NPHP3 mRNA. The mutation leads to activation of a novel cryptic splice site (A), resulting in insertion of 19 bases of intronic sequence in the NPHP3 transcript expressed in the lymphocytes (B).

Figure 6.

Immunohistochemical investigation of nephrocystin-3 in case 1. Some tubules are marked by asterisks. The dysplastic kidney tubules are negative (A), whereas the age-matched control kidney shows distinct cytoplasmic positivity in some tubular segments (B). Scale bars, 50 μm.

Discussion

In two siblings with RHPD whose parents are double fourth cousins, we used a microarray chip to perform a whole-genome single nucleotide polymorphism marker scan to find the genetic locus for the disease. As predicted, only one large chromosomal region (of 21 Mb) appeared to be identical-by-descent. This region contained two known candidate genes, IQCB1 and NPHP3. In the latter, homozygosity for a null mutation was detected. Whereas no clear genotype-phenotype correlation has been observed for mutations in other ciliopathy genes like the CEP290 gene,² such correlation exists for mutations in NPHP3. Null mutations in NPHP3 are reported to cause the lethal condition RHPD with multicystic dysplastic kidneys (Figure 2), whereas less severe mutations cause juvenile nephronophthisis, with few cysts, tubular atrophy, and interstitial fibrosis.¹¹ However, two cases with a juvenile nephronopthisis-like phenotype despite presumed null mutations in NPHP3 have been described, indicating that this correlation may not always be clear-cut. The first case was homozygous for an obligatory splice acceptor-site mutation (IVS26 + 1 G>A), and this child had no extrarenal manifestations of disease.¹² The second case was combined heterozygous for a frameshift mutation (435_438delAAGT) and a splice mutation (IVS24-1G>C) in NPHP3¹³ and was also heterozygous for a frameshift mutation in the NPHP4 gene. This child had liver fibrosis and reached end-stage renal disease at age 3 years. RNA analysis was not reported in these cases. It is possible that at least in the case homozygous for IVS26 + 1 G>A, a partly functional transcript could exist due to alternative splicing and also because the mutation affected the penultimate exon of the gene, possibly resulting in a truncated but somewhat functional protein. The finding of instances with likely triallelic inheritance also adds to the phenotypic heterogeneity in ciliopathies.^13,14 Thus, it is possible that allelic variation or mutations in other genes involved in cilia development and function may affect the phenotype in NPHP3-related disease.

The mutation c.2694-2_2694-1delAG in our ethnic Norwegian family was also reported in a Turkish family in a female fetus and a female sibling who died perinatally (both were homozygous).¹¹ Thus, this may represent a recurrent mutation. Both siblings had enlarged multicystic kidneys, oligohydramnios, and ductal plate malformation. The fetus had normal findings in the pancreas. No information was given about the pancreas in the baby girl that died perinatally. None of the siblings had lateralization defects or polymicrogyria, as seen in our case 2, further demonstrating variability of phenotype in individuals with the same mutation. Polymicrogyria of the cingulate gyrus, as we found in case 2, has not been reported before in RHPD and is probably a rare event in ciliopathies, although it may be underreported. Dixon-Salazar et al¹⁵ found frontal polymicrogyria and thin corpus callosum in three cases with Joubert syndrome with mutations in the AHI1 gene, and they argued that this finding of supratentorial brain affection pointed to a role for this gene also in cortical development. Han et al¹⁶ showed that intact primary cilia and cilia-associated sonic hedgehog signaling are essential for development of adult neural stem cells. Nephrocystin-3 is expressed in neural tissues and interacts with inversin¹¹ and nephrocystin-1,¹² but whether it also interacts with jouberin (the protein encoded by AHI1) is not known. Jouberin, however, interacts with nephrocystin-1.¹⁷

Diagnostic stratification of cystic kidney disease in fetuses and stillborns is challenging because many of the conditions have overlapping clinical pictures. After a thorough clinical and pathological characterization, there may be many candidate genes that could harbor mutation(s). A strategy to find the single major disease-causing gene among many candidates would be useful. In cases where parents are consanguineous and the disease is presumed to be recessive, homozygosity mapping is the method of choice to find the locus (position) of the mutated gene. Most often, such a locus contains one or a few genes known to be involved in a ciliopathy, in other cases, new ciliopathy genes may be searched for. Many ciliopathy genes remain to be discovered, as illustrated by the fact that about half of, for example, Joubert syndrome cases¹⁸ and even more of the Meckel-Gruber syndrome cases,¹⁹ remain genetically unexplained, after all relevant ciliopathy genes have been sequenced. The new high-throughput single nucleotide polymorphism arrays have made homozygosity mapping⁷ feasible also as a routine screening procedure, with an analytic turnaround time of about a week after DNA samples from a sufficient number of family members have been collected.