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An Autosomal Dominant Form of Familial Persistent Hyperinsulinemic Hypoglycemia of Infancy, Not Linked to the Sulfonylurea Receptor Locus¹

Montreal Children’s Hospital Research Institute, Department of Pediatrics, Division of Endocrinology, McGill University (A.K., L.A., C.P.); and the Endocrinology Service, Sainte-Justine Hospital, University of Montreal (C.D.), Montreal, Quebec, Canada

Address all correspondence and requests for reprints to: Constantin Polychronakos, M.D. F.R.C.P., Endocrine Genetics Laboratory, Montreal Children’s Hospital, 2300 Tupper Street, Montreal, Quebec, Canada H3H 1P3. E-mail: mc97{at}musica.mcgill.ca

	Abstract

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Persistent hyperinsulinemic hypoglycemia of infancy (PHHI), a rare disorder due to defective negative feedback regulation of insulin secretion by low glucose levels, is often familial. Most cases are recessively inherited, and mutations of the sulfonylurea receptor gene (SUR) or the closely linked KIR6.2 gene have been found in several families. Both of these genes encode components of the potassium channels responsible for glucose-regulated insulin release. However, in some families recessive PHHI is not linked to the SUR-KIR6.2 locus, suggesting genetic heterogeneity. We report here a French Canadian kindred with hypoglycemia in five first cousins. All five patients had documented hypoglycemia, and all responded well to diazoxide. In two, inappropriately elevated insulin levels during hypoglycemia were documented. This familial clustering strongly suggests the existence of an autosomal dominant form of PHHI. By preliminary linkage analysis, we tested the possibility of a dominant negative SUR or KIR6.2 mutant. The insulin (INS) and glucokinase (GCK) genes were also tested as additional candidates. Microsatellite markers closely linked to each gene were used, and large negative Lod scores were obtained at the known recombination fractions between all three genes studied and the corresponding marker. We conclude that mutation of a gene other than SUR or KIR6.2 is responsible for the dominant PHHI in this family, and this gene cannot be INS or GCK. We propose that a genome-wide search for this gene is important for elucidating this rare disorder and, more importantly, for determining its potential impact on understanding noninsulin-dependent diabetes mellitus and on the effort to develop bioengineered ß-cells for transplantation.

	Introduction

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PERSISTENT hyperinsulinemic hypoglycemia of infancy (PHHI) or neonatal hyperinsulinemia, sometimes referred to as nesidioblastosis, is a disease characterized by severe hypoglycemia starting in the first year of life, with insulin levels inappropriately elevated for the low blood glucose concentration (reviewed in Ref.1). If untreated, it results in hypoglycemic convulsions and the possibility of severe central nervous system sequelae. Diazoxide, the treatment of choice, is effective in the majority of cases, but some patients require partial pancreatectomy (1). The disorder is transient, however, and diazoxide can be discontinued after several months or years of treatment, with an apparent return to normal insulin regulation (1). This is a rare phenotype whose frequency in a noninbred Caucasian population is estimated at 1:50,000 (2). Although most cases are sporadic, a considerable proportion [5 of 24 in 1 study (3)] are familial, inherited as autosomal recessive (3, 4, 5). Segregation analysis shows that recessive inheritance can probably explain most, if not all, sporadic cases (3).

Familial recessive PHHI was recently found to be due to homozygous mutations of the high affinity sulfonylurea receptor gene (SUR) (6) or of the closely linked and functionally related KIR6.2 gene (7), both mapping on 11p15.1. Both genes encode components of ATP-dependent potassium channels involved in glucose-regulated insulin release (8, 9). Closure of these channels by binding of sulfonylurea compounds to SUR or their inactivation by mutation causes hyperinsulinemia (8).

Although recessive inheritance can explain most of the familial occurrence of PHHI, there is evidence of genetic heterogeneity. Vertical transmission in the absence of consanguinity has been reported in six patients from two families, implying a dominantly inherited form (10). In this paper we report a French Canadian kindred in which PHHI is clearly inherited as a dominant trait. Although the different mode of inheritance suggests the involvement of a different gene, the possibility of a dominant negative SUR mutation must be ruled out. Other possibilities, arising from the known physiology of the ß-cell, would be an alteration in the regulatory sequences of the insulin gene (INS) interfering with the normal shut-down of the gene in the presence of low glucose or a gain of function in the glucokinase gene (GCK), the first step in the glucose-sensing process in the ß-cell. The purpose of the work reported here was to test these possibilities by linkage analysis.

	Subjects and Methods

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Subjects

The pedigree is shown in Fig. 1. All five affected first cousins in generation III had seizures in infancy, and four of five had documented hypoglycemia (blood glucose <2 mmol/L while symptomatic). Patient III-1, the first to be ascertained, was also included even though his old files containing the glucose values on which the diagnosis was based could not be located. He was investigated in a university hospital, diagnosed as having hyperinsulinism, and successfully treated with diazoxide. The diagnosis could not have been biased by the family history, as he was the first case to be ascertained. All five patients responded well to treatment with diazoxide, and recurrences upon attempts at discontinuation were documented in two cases. Full documentation of hyperinsulinemia in the face of hypoglycemia was found for two of them (Table 1). Two more had no ketonuria while hypoglycemic, strongly suggesting hyperinsulinemia. No other cause of hypoglycemia or neurological symptoms could be identified in any family member. These five cases will be referred to as documented PHHI.

View larger version (28K): [in this window] [in a new window]   Figure 1. Pedigree of the kindred exhibiting autosomal dominant inheritance of PHHI. Biochemical documentation was available only for the individuals in generation III. Only these subjects and their common grandfather were designated affected for the purpose of the linkage analysis.

Historical information from previous generations strongly corroborates dominant inheritance. The common grandfather of all cases (I-4), who at the age of 79 yr is clinically normal, had a history of unexplained seizures early in life, as did his 3 siblings and 6 of his 12 children, 2 of whom are parents of documented PHHI cases. Seven of the 10 clinically suspected but undiagnosed and untreated individuals in the earlier generations died early in life or had severe neurological sequelae preventing them from having children.

Methods

Lymphoblastoid cell lines were established from blood samples taken with informed consent from the individuals whose genotypes are shown in Fig. 1. The D11S902 simple sequence repeat, mapped to chromosome 11p15.1, was used to evaluate linkage to the SUR-KIR6.2 locus, as it has been mapped to a position centromeric to SUR, at a distance of less than 0.8 centimorgans (cM) from SUR (11). To evaluate linkage to INS, we chose the tyrosine hydroxylase tetranucleotide repeat (HUMTHO1), located within a few kilobases of the INS promoter (12). Finally, as our family was noninformative for the GCK microsatellite described by Tanizawa et al. (13), we used the D7S478 marker, mapped to chromosome 7p, 5 cM centromeric to GCK (Genome Data Base map C7 M58).

All markers were genotyped by PCR, with primers and amplification protocols obtained from Research Genetics (Huntsville, AL). The MLINK module of the LINKAGE software package, provided by Dr. Jurg Ott, was used for two-locus linkage analysis (14). It calculates the Lod score, which is the decimal logarithm of the ratio of the likelihood that the observed cosegregation of marker alleles and disease status would be seen in the presence of linkage, over the likelihood that it would be seen in the absence of linkage. Lod scores are calculated for several different assumed distances between marker and disease locus, expressed by , the recombination fraction (the probability that recombination will occur over that distance in each meiosis). A Lod score of 3 or more (1000-fold higher likelihood) constitutes proof of linkage, whereas a score less than -3 (1000-fold lower) eliminates the locus. The value at which the maximal score is obtained is an indication of genetic distance between disease and marker.

For calculation of the Lod score, only the five documented cases and their common grandfather were assigned affected status. Unknown affection status was assigned to all neurologically affected but biochemically undocumented subjects in generation II. Our analysis assumed a priori a dominant model with 90% penetrance. Analysis under a deliberate underestimate of penetrance at 50% gave essentially the same results, as most of the genetic information in our pedigree was derived from affected individuals and obligate carriers.

	Results

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The genotypes of all individuals tested are shown in Fig. 1. SUR and INS can be eliminated using information from only the biochemically documented cases; individuals III-10 and III-14, both with documented hyperinsulinemia, share no D11S902 alleles. The only HUMTH01 allele shared by all documented cases is 4, but it is not identical by descent, being grand-maternal in III-1 and grand-paternal in III-10 and III-14.

Recombinations were also found with the GCK-linked marker D7S478, but because of the considerable genetic distance this candidate gene was ruled out by a negative Lod score at a value of 0.05, corresponding to the known genetic distance of this marker from GCK (Table 2).

	Discussion

Top Abstract Introduction Subjects and Methods Results Discussion References

Based on the number of affected individuals, the penetrance of the hypoglycemia phenotype in our pedigree is high, although it cannot be 100%, as two of the parents of documented PHHI cases gave no history of clinical manifestations. This apparent skipping of a generation may appear paradoxical given the otherwise high penetrance. It should be borne in mind, however, that 7 of the 10 individuals who were probably affected clinically but not diagnosed or treated in early life were prevented from reproducing by early death or severe neurological sequelae, which may result in a bias in favor of unaffected obligate carriers, who are parents of any given number of ascertained cases. In addition, nutritional or other environmental factors might have influenced the appearance of the phenotype in a generation-specific manner. Metabolic studies on the surviving members of the first and second generations might shed light on this question.

Regardless of whether the gene responsible for dominant PHHI is currently unknown or is one already known to be involved in ß-cell function, the importance of identifying it is not limited to elucidating this unusual form of a rare disorder. Identification of a gene involved in the regulation of insulin secretion based on ambient glucose is likely to contribute to better understanding of type 2 diabetes, a disease due to a compromised compensatory increase in insulin secretion in the face of insulin resistance. Already the identification of SUR as the gene involved in PHHI has spurred genetic studies showing type 2 diabetes linkage to SUR (16). Another endeavor that was given an enormous boost by the discovery of SUR and will directly benefit from the study of other genes involved in ß-cell regulation by glucose is the development of bioengineered ß-cells for the treatment of insulin-dependent diabetes (17). For these reasons, we believe that a genome-wide search for the dominant PHHI gene must be given high priority.

	Acknowledgments

 
We thank the following physicians who have treated the patients studied and provided valuable information: J. Letarte, C. Gervais, S. Woods, A. Schiffrin, and P. Crock. We also thank Dr. Guy Rouleau for technical support. Finally, we thank N. Giannoukakis for contribution to the initial planning of this study.

	Footnotes

 
¹ This work was supported by the Canadian Diabetes Association.

² Recipient of the McGill University Alan Ross Academic Fellowship.

Received November 4, 1996.

Revised January 3, 1997.

Accepted January 13, 1997.

	References

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